Methods for detecting epigenetic modifications

ABSTRACT

A method of detecting a predisposition to, or the incidence of, cancer in a sample comprises detecting an epigenetic change in at least one gene selected from an NDRG4/NDRG2 subfamily gene, GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3, wherein detection of the epigenetic change is indicative of a predisposition to, or the incidence of, cancer. Also described are pharmacogenetic methods for determining suitable treatment regimens for cancer and methods for treating cancer patients, based around selection of the patients according to the methods of the invention. The present invention is also concerned with improved methods of collecting, processing and analyzing samples, in particular body fluid samples. These methods may be useful in diagnosing, staging or otherwise characterizing various diseases. The invention also relates to methods for identifying, diagnosing, staging or otherwise characterizing cancers, in particular gastrointestinal cancers such as colorectal cancers, gastric cancers and oesophageal cancers. The methods of the invention relate, inter alia, to isolating and analyzing the human DNA component from faecal samples and blood-based samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/971,461, filed May 4, 2018, which is a continuation of U.S.patent application Ser. No. 14/613,574, filed Feb. 4, 2015, now U.S.Pat. No. 9,982,308, which is a continuation of U.S. patent applicationSer. No. 12/522,648, filed Jan. 6, 2010, now U.S. Pat. No. 8,969,046,which is a § 371 U.S. National Entry of PCT/GB2008/000056, filed Jan. 9,2008, which claims the priority benefit of U.S. Provisional PatentApplication Nos. 60/978,261, filed Oct. 8, 2007; 60/960,130, filed Sep.17, 2007; and 60/879,332, filed Jan. 9, 2007, each of which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and kits for identifying anddiagnosing cancer which include detecting an epigenetic change, such asa change in the methylation status, or the expression levels, or acombination thereof of any one or more of a number of genes. Alsodescribed are pharmacogenetic methods for determining suitable treatmentregimens for cancer and methods for treating cancer patients, basedaround selection of the patients according to the methods of theinvention. The present invention is also concerned with improved methodsof collecting, processing and analyzing samples, in particular bodyfluid samples. More particularly, the invention relates to methods foridentifying epigenetic changes in body fluid samples. These methods maybe useful in diagnosing, staging or otherwise characterizing variousdiseases. The invention also relates to methods for identifying,diagnosing, staging or otherwise characterizing cancers, in particulargastrointestinal cancers such as colorectal cancers, gastric cancers andoesophageal cancers. The methods of the invention relate, inter alia, toisolating and analyzing the human DNA component from faecal samples andblood-based samples.

BACKGROUND OF THE INVENTION

In their earliest stages most cancers are clinically silent. Patientdiagnosis typically involves invasive procedures that frequently lacksensitivity and accuracy. Highly reliable, non-invasive screeningmethods would permit easier patient screening, diagnosis and prognosticevaluation.

Tumour derived markers are biological substances that are usuallyproduced by malignant tumours. Ideally a tumour derived marker should betumour-specific, provide an indication of tumour burden and should beproduced in sufficient amounts to allow the detection of minimaldisease. Most tumour derived markers used in clinical practice aretumour antigens, enzymes, hormones, receptors and growth factors thatare detected by biochemical assays. The detection of DNA alterationssuch as mutations, deletions and epigenetic modifications (Baylin etal., 2000) provide another means for identifying cancers.

An epigenetic modification can be described as a stable alteration ingene expression potential that takes place during development and cellproliferation, mediated by mechanisms other than alterations in theprimary nucleotide sequence of a gene. It is now general knowledge thatboth genetic and epigenetic alterations can lead to gene silencing andcellular dysfunction. Synergy between these two processes drives tumorprogression and malignancy. Three related mechanisms that causealteration in gene expression are recognised: DNA methylation, histonecode changes and RNA interference.

DNA hypermethylation is an epigenetic modification whereby the geneactivity is controlled by adding methyl groups (CH3) to specificcytosines of the DNA. In particular, methylation occurs in the cytosineof the CpG dinucleotides (CpG islands) which are concentrated in thepromoter regions and introns in human genes (P. A. Jones et al., 2002;P. W. Laird et al., 2003). Methylation is associated with genesilencing. DNA hypermethylation is found to be involved in a variety ofcancers including lung, breast, ovarian, kidney, cervical, prostate andalso colorectal cancer. Methylation patterns of DNA from cancer cellsare significantly different from those of normal cells. Therefore,detection of methylation patterns in appropriately selected genes ofcancer cells can lead to discrimination of cancer cells from normalcells, thereby providing an approach to early detection of cancer.

DNA tumour markers, in particular DNA methylation markers, offer certainadvantages when compared to other biochemical markers. An importantadvantage is that DNA alterations often precede apparent malignantchanges and thus may be of use in early diagnosis of cancer. Since DNAis much more stable and, unlike protein, can be amplified by powerfulamplification-based techniques for increased sensitivity, it offersapplicability for situations where sensitive detection is necessary,such as when tumour DNA is scarce or diluted by an excess of normal DNA(Sidransky et al., 1997). Bodily fluids provide a cost-effective andearly non-invasive procedure for cancer detection. In this context,faecal-based cancer testing has been one area of investigation.

Human colorectal cancer has provided a good model for investigatingwhether DNA cancer markers can be adopted as an optimal faecal-baseddiagnostic screening test. Central to faecal-based colorectal cancertesting has been the identification of specific and sensitive cancerderived markers.

The N-Myc downstream-regulated gene (NDRG) family comprises four familymembers: NDRG1 (NDRG-family member 1), NDRG2 (NDRG-family member 2),NDRG3 (NDRG-family member 3) and NDRG4 (NDRG-family member 4). The humanNDRG1 and NDRG3 belong to one subfamily, and NDRG2 and NDRG4 to another.At amino acid (aa) level, the four members share 53-65% identity. Thefour proteins contain an alpha/beta hydrolase fold as in human lysosomalacid lipase but are suggested to display different specific functions indistinct tissues.

NDRG1 codes for a cytoplasmic protein believed to be involved in stressresponses, hormone responses, cell growth, and cell differentiation.NDRG1 has been demonstrated to be upregulated during celldifferentiation, repressed by N-myc and c-myc in embryonic cells, andsuppressed in several tumor cells (Qu X et al., 2002; Guan et al.,2000).

NDRG3 is believed to play a role in spermatogenesis since it is highlyexpressed in testis, prostate and ovary (Zhao W et al., 2001). Itsinvolvement in brain cancer development has also been suggested (Qu X etal. 2002).

NDRG2 codes for a cytoplasmic protein that seems to be involved inneurite outgrowth and in glioblastoma carcinogenesis (Deng Y et al.,2003). It is upregulated at both the RNA and protein levels inAlzheimer's disease brains (Mitchelmore C et al., 2004), and has alsobeen suggested to play an important role in the development of braincancer (Qu X et al. 2002), pancreatic cancer and liver cancer (Hu X L etal., 2004).

The NDRG4 cytoplasmic protein is involved in the regulation of mitogenicsignalling in vascular smooth muscles cells (Nishimoto S et al). TheNDRG4 gene contains 17 exons, and several alternatively splicedtranscript variants of this gene have been described. NDRG4 may also beinvolved in brain cancer development (Qu X et al. 2002).

Suppressed expression of NDRG-family genes has been demonstrated in anumber of tumours (Qu X et al. 2002) and the involvement of DNA promoterhypermethylation is limited to the reporting of NDRG2 methylation inbrain tumors (Lusis et al., 2005).

Initially, faecal-based DNA assays investigated the usefulness ofspecific point mutations markers for detecting colorectal cancer. Later,the DNA integrity in faecal samples proved to be a useful marker(Boynton et al., 2003). Finally, faecal testing based on DNA alterationsgradually evolved into the development of a multi-target DNA assay usingspecific point mutation markers, a microsatellite instability marker anda marker for DNA integrity.

Recently, the potential of faecal DNA testing targeting epigeneticalterations has been investigated (Müller et al., 2004, Chen et al.,2005) and has been added to the multi-target DNA assay. Genes having analtered methylation status traceable in faecal DNA from colon cancerpatients versus control samples from healthy subjects have beendiscovered (Belshaw et al., 2004; Petko et al., 2005; Lenhard et al.,2005; Miller et al., 2004; Chen et al., 2005 and Lueng et al., 2004).

Factors that may influence the sensitivity of the selected markers aresampling processing procedures and DNA isolation and extractionprotocols. One challenge faced by researchers investigating colorectalcancer is the diversity of DNA present in stool samples. Most of the DNArecovered from faecal samples is bacterial in origin, with the human DNAcomponent representing only a very small minority. Human DNA from cellssloughed from the colonic mucosa represents as little as 0.1 to 0.01% ofthe total DNA recoverable from stool. Additionally, the human DNArecovered is highly heterogeneous. Normal cells are sloughed into thecolonic lumen along with only a small amount of tumour cells(approximately 1% of the cells sloughed). Thus, the DNA of interestrepresents only a very small percentage of the total DNA isolated fromstool. Therefore, along with the exploration of suitable DNA markers,techniques for improved DNA isolation and enrichment of the human DNAcomponent from faecal samples have been developed for more sensitivecancer detection.

The initial DNA isolation techniques typically recovered DNA from 10 gto 4 g stool and more conveniently purified the human DNA componentusing streptavidin-bound magnetic beads (Dong et al., 2001; Ahlquist etal., 2000). Further improvements in recovery of target human DNA fromstool comprised an electrophoresis-driven separation of target DNAsequences, using oligonucleotide capture probes immobilized in anacrylamide gel (Whitney et al., 2004). Later, when DNA integrity provedto be a suitable marker it was also important to prevent degradationduring sample handling. Improved results were obtained with stoolsamples frozen as quickly as possible after collection. Alternatively,stabilization buffer was added to the stool samples before furthertransport (Olson et al., 2005). A recent improvement involves the use ofan MBD column to extract methylated human DNA in a high background offecal bacterial DNA (Zou et al., 2007). However, despite these advances,current tools for cancer detection in faecal samples are stillunsatisfactory.

Cancer at its early stage may release its cells or free DNA into bloodthrough apoptosis, necrosis or local angiogenesis, which establishes abasis for blood-based cancer testing. The usefulness of DNA methylationmarkers for detecting colorectal cancers in serum and plasma has beendemonstrated (Grady et al., 2001, Leung et al., 2005; Nakayama et al.,2007). However, the potential use of serum and plasma for cancerdetection is hampered by the limited level of methylated DNA present inthe total DNA collected from plasma and serum samples (Zou et al. (2002)Clin Cancer Res 188-91). A further drawback is the partial degradationof the methylated DNA due to bisulfite treatment, a treatment steprequired by many techniques that monitor DNA methylation.

Methods and compositions for detection of early colorectal cancer orpre-cancer using blood and body fluids have been described.

WO 2006/113770 describes methods in which samples are pooled andconcentrated in an attempt to maximize DNA input per reaction. Theinitial processing of 45 ml of blood allowed a median DNA recovery of3.86 ng/ml plasma. This was shown to result in a sensitivity of 57% andspecificity of 96% for detection of colorectal cancer using a specificreal-time assay for detecting whether The Septin 9 gene was methylated.Bisulphite treatment was focused on large volume treatment and achievingmaximal conversion.

Lofton-Day et al. (AACR general meeting April 2007, Los Angeles, USA)mention improved detection of colorectal cancer, and obtained a 70%sensitivity and 90% specificity, with the same marker (Septin 9). Theproposed method utilised four blood draws (40 ml blood), doublecentrifugation for plasma recovery and required four PCR reactions to becarried out for each sample tested. Three out of the four reactions usedinput DNA equivalent to 2 ml of plasma per PCR reaction. The fourthreaction used a 1/10 dilution of this input DNA. Thus, repeated assayswere required (at least 4) and an algorithm utilised to determine thefinal result. A sample was deemed positive if either two out of thethree reactions with input DNA equivalent to 2 ml of plasma, or thediluted measurement, were positive for the Septin 9 assay. The improvedsensitivity by using the diluted samples indicates the presence ofinhibitors in the methods, a phenomena also described by Nakayama et al.(2007, Anticancer Res. 27(3B):1459-63).

The processing of smaller amounts of blood have been described as well(US 20070141582, Hong-Zhi Zou et al., and Satoru Yamaguchi et al) butall result in low level of methylated modified DNA detection.

Thus, current blood-based screening methods lack sensitivity.

SUMMARY OF THE INVENTION

The invention, as set out in the claims, is based around the findingthat NDRG4/2 subfamily genes, undergoe CpG island promotermethylation-associated gene silencing in human cancer cells, inparticular colon cancer cells. The hypermethylation of the NDRG familygene, such as NDRG4 and/or NDRG2, in particular in the promoter regionleads to its loss of expression. Importantly, the presence of aberrantmethylation at the NDRG4/2 subfamily gene promoter has a prognosticvalue. The epigenetic loss of NDRG4/2 function can be rescued by the useof DNA demethylating agents and thus provides for a method fortreatment. These findings underline the significance of the epigeneticsilencing of the NDRG4/2 subfamily genes as one key step in cancerdevelopment and may have an important clinical impact for the treatmentof the patients.

The present invention is also based upon the discovery of specific genesand panels of genes whose methylation status is linked to the incidenceof, or predisposition to, gastrointestinal cancers such as colorectalcancer. Use of these genes for detecting gastrointestinal cancers suchas colorectal cancer, in particular in the context of appropriate tissueor faecal (stool) samples or of appropriate blood samples (orderivatives thereof) respectively, has been shown to produce highlysensitive and specific results. The invention provides also for a methodfor isolating increased amount of DNA from faecal samples, which resultsin improved sensitivity of detection of colorectal cancer in faecalsamples.

The invention also provides a method for determining the methylationstatus of a gene of interest in a blood based sample, which requiresonly low volumes of blood sample equivalent to generate specific andsensitive results. This is advantageous since it permits smaller bloodsamples to be obtained from the subject under test.

Accordingly, in a first aspect, the invention provides a method ofdetecting a predisposition to, or the incidence of, cancer in a samplecomprising detecting an epigenetic change in at least one gene selectedfrom an NDRG4/NDRG2 subfamily gene (in particular NDRG4), GATA4, OSMR,GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3 and JAM3, wherein detection of the epigeneticchange is indicative of a predisposition to, or the incidence of,cancer.

Subsets of genes for all aspects and embodiments of the inventioninclude an NDRG4/NDRG2 subfamily gene (in particular NDRG4), GATA4,OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC and MGMT and TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 respectively. Each subset may beparticularly applicable to bodily fluid samples, such as stool andplasma samples as discussed herein.

By “epigenetic change” is meant a modification in the gene caused by anepigenetic mechanism, such as a change in methylation status or histoneacetylation for example. Frequently, the epigenetic change will resultin an alteration in the levels of expression of the gene which may bedetected (at the RNA or protein level as appropriate) as an indicationof the epigenetic change. Often the epigenetic change results insilencing or down regulation of the gene, referred to herein as“epigenetic silencing”. The most frequently investigated epigeneticchange in the methods of the invention involves determining themethylation status of the gene, where an increased level of methylationis typically associated with the relevant cancer (since it may causedown regulation of gene expression).

In a related aspect, the invention provides a method of diagnosingcancer or predisposition to cancer comprising detecting epigeneticsilencing of the NDRG4/NDRG2 subfamily gene, wherein epigeneticsilencing of the gene is indicative for cancer or predisposition tocancer.

The NDRG family genes have been characterised in the art (see, forexample, Qu X et al., 2002 and references cited therein) and theirepigenetic silencing can be assessed in terms of DNA methylation statusor expression levels as determined by their methylation status.

In one embodiment, the invention provides for a method of diagnosingcancer or predisposition to cancer comprising detecting epigeneticsilencing of the NDRG4/NDRG2 subfamily gene, wherein epigeneticsilencing of the NDRG2/NDRG4-family gene is detected by determination ofthe methylation status of the NDRG4/2 family gene and whereinmethylation of the gene is indicative for cancer or predisposition tocancer.

Since methylation of the NDRG4/NDRG2 subfamily gene manifests itself inreduced expression of the gene the invention also provides for a methodof diagnosing cancer or predisposition to cancer comprising detectingepigenetic silencing of the NDRG4/NDRG2 subfamily gene, whereinepigenetic silencing of the NDRG2/NDRG4-family gene is determined bymeasurement of expression levels of the gene, wherein reduced expressionof the gene is indicative for cancer or predisposition to cancer.

In a related aspect, the invention provides method of prognosis tocancer or predisposition to cancer comprising detecting epigeneticsilencing of the NDRG4/NDRG2 subfamily gene, wherein epigeneticsilencing of the gene is indicative for cancer development orpredisposition to cancer. Preferably, epigenetic silencing is detectedby determination of the methylation status and/or measurement ofexpression levels of the NDRG2/NDRG4-family gene.

The invention also provides a method of detecting a predisposition to,or the incidence of, cancer and in particular a gastrointestinal cancersuch as colorectal cancer in a sample comprising detecting an epigeneticchange in at least one gene selected from GATA4, OSMR, NDRG4, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, and MGMT, and/or TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3 and JAM3, wherein detection of the epigeneticchange is indicative of a predisposition to, or the incidence of, cancerand in particular a gastrointestinal cancer such as colorectal cancer.These subsets of genes may be particularly useful where faecal testsamples are utilised (and plasma in certain embodiments).

In a related aspect, the invention also provides a method of detecting apredisposition to, or the incidence of, cancer and in particular agastrointestinal cancer such as colorectal cancer in a sample and inparticular in a blood sample, or derivative thereof comprising detectingan epigenetic change in at least one gene selected from GATA4, OSMR,NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (together with any suitable subsetor panel thereof), wherein detection of the epigenetic change isindicative of a predisposition to, or the incidence of, cancer and inparticular a gastrointestinal cancer such as colorectal cancer.

By “NDRG2/NDRG4 subfamily gene” is meant any gene which is taken fromthe subfamily to which NDRG4 and NDRG2 belong and includes according toall aspects of the invention NDRG2 and NDRG4. Note that “NDRG1, NDRG2,NDRG3 and NDRG4” is the standard nomenclature approved by the humangenome organisation for the NDRG family genes, to ensure that eachsymbol is unique. The listed accession number for these genes can befound at www.gene.ucl.ac.uk/nomenclature.

NDRG family genes encompass not only the particular sequences found inthe publicly available database entries, but also encompass transcriptvariants of these sequences. Variant forms of the encoded proteins maycomprise post-translational modification, may result from splicedmessages, etc. . . . NDRG4 has transcript variants having the accessionnumbers NM_020465 and NM_022910. NDRG2 has several transcript variantshaving the accession numbers, NM_201535, NM_201536, NM_201537,NM_201538, NM_201539, NM_201540, NM_2015401 and NM_016250. Variantsequences may have at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity to sequences in the database entries orsequence listing. Computer programs for determining percent identity areavailable in the art, including Basic Local Alignment Search Tool(BLASTS) available from the National Center for BiotechnologyInformation.

GATA4 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 8 (location p23.1-p22) andthe gene sequence is listed under the accession numbers AK097060,NM_002052 and ENSG00000136574. The gene encodes GATA binding protein 4.

OSMR is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 5 (location p13.2) and thegene sequence is listed under the accession numbers U60805, NM_003999and ENSG00000145623. The gene encodes oncostatin M receptor.

NDRG4 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 16 (location q21-q22.3) andthe gene sequence is listed under the accession numbers AB044947 andENSG00000103034. The gene encodes NDRG family member 4.

GATA5 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 20 and the gene sequence islisted under the accession number ENSG00000130700. The gene encodes GATAbinding protein 5.

SFRP1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 8 (location p11.21) and thegene sequence is listed under the accession numbers AF017987, NM_003012and ENSG00000104332. The gene encodes secreted frizzled-related protein1.

ADAM23 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 2 (location q33) and thegene sequence is listed under the accession numbers AB009672 andENSG00000114948. The gene encodes ADAM metallopeptidase domain 23.

JPH3 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 16 (location q24.3) and thegene sequence is listed under the accession numbers AB042636 andENSG00000154118. The gene encodes junctophilin 3.

SFRP2 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 4 (location q31.3) and thegene sequence is listed under the accession numbers AF017986 andENSG00000145423. The gene encodes secreted frizzled-related protein 2.

APC is the gene symbol approved by the HUGO Gene Nomenclature Committee.The gene is located on chromosome 5 (location q21-q22) and the genesequence is listed under the accession numbers M74088 andENSG00000134982. The gene encodes adenomatosis polyposis coli.

The MGMT gene encodes 06-methylguanine-DNA methyltransferase (MGMT),which is a cellular DNA repair protein that rapidly reverses alkylation(e.g. methylation) at the 06 position of guanine, thereby neutralizingthe cytotoxic effects of alkylating agents such as temozolomide (TMZ)and carmustine (1-3). MGMT is the gene symbol approved by the HUGO GeneNomenclature Committee. The gene is located on chromosome 10 (location10q26) and the gene sequence is listed under the accession numbersM29971, NM_002412 and ENSG00000170430.

BNIP3 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 10 (location 10q26.3) andthe gene sequence is listed under the accession numbers U15174 andENSG00000176171. The gene encodes the BCL2/adenovirus E1B 19 kDainteracting protein 3.

FOXE1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 9 (location 9q22) and thegene sequence is listed under the accession numbers U89995 andENSG00000178919. The gene encodes the forkhead box E1 (thyroidtranscription factor 2)

JAM3 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 11 (location 11q25) and thegene sequence is listed under the accession numbers AF356518, NM_032801and ENSG00000166086. The gene encodes the junctional adhesion molecule3.

PHACTR3 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 20 (location 20q13.32) andthe gene sequence is listed under the accession numbers AJ311122,NM_080672 and ENSG00000087495. The gene encodes the phosphatase andactin regulator 3.

TFPI2 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 7 (location 7q22) and thegene sequence is listed under the accession numbers L27624 andENSG00000105825. The gene encodes the tissue factor pathway inhibitor 2.

SOX17 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 8 (location 8q11.23) andthe gene sequence is listed under the accession numbers AB073988 andENSG00000164736. The gene encodes the SRY (sex determining region Y)-box17.

SYNE1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 6 (location 6q25) and thegene sequence is listed under the accession numbers AB018339 andENSG00000131018. The gene encodes the spectrin repeat containing,nuclear envelope 1.

Of course, as appropriate, the skilled person would appreciate thatfunctionally relevant variants of each of the gene sequences may also bedetected according to the methods of the invention. For example, themethylation status of a number of splice variants may be determinedaccording to the methods of the invention. Variant sequences preferablyhave at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% nucleotide sequence identity with the nucleotide sequences in thedatabase entries. Computer programs for determining percentagenucleotide sequence identity are available in the art, including theBasic Local Alignment Search Tool (BLAST) available from the NationalCenter for Biotechnology Information.

The methods of the invention are generally ex vivo or in vitro methodscarried out on a test sample, in particular on an isolated test sample.The methods can be used to diagnose any suitable type of cancer. Thecancer comprises, consists essentially of or consists of a neoplasia ofthe gastrointestinal tract such as gastrointestinal cancer in oneembodiment. In specific embodiments, the methods of the invention areapplied to colorectal cancer, gastric cancer and/or oesophageal cancer.In more specific embodiments, the methods are used to diagnosecolorectal cancer, and more particularly to diagnose hereditarynonpolyposis colon cancer and/or sporadic colorectal cancer.Alternatively, the methods are aimed at diagnosis of gastric cancer.Preferably, the methods are used to diagnose colorectal cancer and/orgastric cancer. The methods may be used to detect carcinoma or adenoma,in particular advanced adenoma. The methods may be employed in thediagnosis of both diffuse type and intestinal type carcinomas of thestomach, particularly when the methylation status of NDRG4 isdetermined. In one embodiment the methods may also include the step ofobtaining the sample.

In one specific embodiment, the methods are used to diagnose oesophagealadenocarcinoma. In particular, the methylation status of the NDRG4 gene(promoter) has been shown for the first time herein to be linked withhigh sensitivity and specificity to the incidence of this particularcancer type. Oesophageal adenocarcinoma may be distinguished fromoesophageal squamous cell carcinomas on this basis.

The “test sample” can be any tissue sample or body fluid. Preferably,the test sample is obtained from a human subject. In specificembodiments, the sample is taken from the gastrointestinal tract. Thesample may be a colorectal tissue sample or a colon, rectal,oesophageal, stomach or appendix tissue sample or a faecal or bloodbased sample from a subject. For faecal samples the methods arepreferably used with respect to detecting gastrointestinal cancers suchas colorectal cancer as discussed herein, but may also be useful inidentifying potentially dangerous adenomas. Different markers and panelsof markers may be most useful with a specific sample type, such as atissue, blood based or faecal sample as discussed herein in detail.

Thus, for example, in one embodiment, the methods of the inventioninvolve detecting an epigenetic change, and in particular determiningthe methylation status, of (at least) the NDRG4 gene in a faecal testsample, wherein detection of the epigenetic change, in particular(hyper)methylation of the NDRG4 gene (promoter) is indicative ofgastrointestinal neoplasias/cancer, in particular colorectal cancer,such as adenomas and carcinomas, gastric cancer and otheradenocarcinomas of the gastrointestinal tract (such as oesophagealadenocarcinoma) and/or diffuse type and intestinal type carcinomas ofthe stomach.

The subject may be suspected of being tumorigenic. More specifically thesubject may be suspected of suffering from a cancer, such as agastrointestinal cancer and in particular colorectal cancer, asdiscussed herein. However, any other suitable test samples in whichepigenetic silencing of the appropriate gene or genes of the invention,for example an NDRG4/NDRG2 subfamily gene, can be determined to indicatethe presence of cancer are included within the scope of the invention.Preferred panels and subsets of genes are presented herein which providesensitive and specific diagnosis, including early stage detection, of agastrointestinal cancer such as colorectal cancer based upon appropriatesamples such as tissue, faecal and plasma samples as discussed herein.Thus, in embodiments in which tissue samples are utilised, the methodsof the invention may comprise, consist essentially of or consist ofdetecting an epigenetic change in a panel of genes comprising OSMR,GATA4 and ADAM23 or OSMR, GATA4 and GATA5, wherein detection of theepigenetic change in at least one of the genes in the panel isindicative of a predisposition to, or the incidence of, agastrointestinal cancer such as colorectal cancer. The tissue sample maycomprise, consist essentially of or consist of a colon and/or rectaland/or appendix sample for example.

Other DNA-containing sample which may be used in the methods of theinvention include samples for diagnostic, prognostic, or personalisedmedicinal uses. These samples may be obtained from surgical samples,such as biopsies or fine needle aspirates, from paraffin embeddedtissues, from frozen tumour tissue samples, from fresh tumour tissuesamples or from a fresh or frozen body fluid, for example. Non-limitingexamples include whole blood or parts/fractions thereof, bone marrow,cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph fluid,serum, plasma, urine, chyle, ejaculate, sputum, nipple aspirate, saliva,swabs specimens, colon wash specimens and brush specimens. The tissuesand body fluids can be collected using any suitable method, many suchmethods are well known in the art. Assessment of a paraffin-embeddedspecimen can be performed directly or on a tissue section. Tissuesamples are generally taken from the tissue suspected of beingtumourigenic.

In a specific embodiment, the test sample is a blood sample. Any bloodsample, or derivative thereof may be utilised. The blood sample, orderivative thereof may comprise, consist essentially or whole blood orany suitable DNA containing parts/fractions thereof. In specificembodiments, the blood sample or derivative thereof comprises, consistessentially of or consists of serum or plasma. The blood sample may becollected using any suitable method, many such methods are well known inthe art. In one embodiment, the methods of the invention alsoincorporate the step of obtaining the blood sample. Any appropriateblood sample may be utilised in the methods of the invention, providedit contains sufficient DNA.

In a specific embodiment, the volume of the blood sample, or derivativethereof that is utilised in the methods is around 5 to 15 ml, such as 10ml.

Blood samples, or derivatives thereof, may be stored prior to use in themethods of the invention once obtained. They may be frozen for exampleat a suitable temperature, such as around −80° C.

It is preferred that the blood sample, or derivative thereof comprises,consists essentially of or consists of a plasma or serum sample. Plasmamay be derived from whole blood by any suitable means. In oneembodiment, the plasma sample is obtained by centrifugation of wholeblood. Centrifugation may be carried out at any suitable speed and forany suitable period of time and under any suitable conditions as may bedetermined by one skilled in the art. For example, centrifugation may becarried out at between around 1000 and 3000 g. Centrifugation may becarried out for between around 1, 2, 3, 4, or 5 and 10, 11, 12, 13, 14or 15 minutes for example. Centrifugation may be carried out at lowtemperatures, such as between around 0 and 5° C., for example 4° C., tomaintain integrity of the sample. Multiple centrifugation steps may beemployed in order to obtain the plasma sample. In a specific embodiment,two centrifugation steps are employed to obtain the plasma sample.

In embodiments where blood and in particular plasma or serum samples areutilised, the at least one gene may be selected from OSMR, SFRP1, NDRG4,GATA5, ADAM23, JPH3, SFRP2 and APC. As shown below, these genes providesensitive and specific methods for diagnosing colorectal cancer inplasma samples. Suitable panels in this context comprise, consistessentially of or consist of OSMR, NDRG4, GATA5 and ADAM23. Additionalgenes which may be employed in plasma or serum based methods includeTFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3, and JAM3.

“Diagnosis” is defined herein to include screening for a disease orpre-indication of a disease, identifying a disease or pre-indication ofa disease, monitoring the staging and the state and progression of thedisease, checking for recurrence of disease following treatment andmonitoring the success of a particular treatment. The methods of theinvention may also have prognostic value, and this is included withinthe definition of the term “diagnosis”. The prognostic value of themethods of the invention may be used as a marker of potentialsusceptibility to a number of gastrointestinal cancers such ascolorectal cancer or as a marker for progression from adenoma to cancerfor example. Thus patients at risk may be identified before the diseasehas a chance to manifest itself in terms of symptoms identifiable in thepatient.

The methods of the invention may be carried out on purified orunpurified DNA-containing samples. However, in specific embodiments, DNAis isolated/extracted/purified from the sample. Any suitable DNAisolation technique may be utilised. Examples of purification techniquesmay be found in standard texts such as Molecular Cloning—A LaboratoryManual (Third Edition), Sambrook and Russell (see in particular Appendix8 and Chapter 5 therein). In one embodiment, purification involvesalcohol precipitation of DNA. Preferred alcohols include ethanol andisopropanol. Suitable purification techniques also include salt-basedprecipitation methods. Thus, in one specific embodiment the DNApurification technique comprises use of a high concentration of salt toprecipitate contaminants. The salt may comprise, consist essentially ofor consist of potassium acetate and/or ammonium acetate for example. Themethod may further include steps of removal of contaminants which havebeen precipitated, followed by recovery of DNA through alcoholprecipitation.

In an alternative embodiment, the DNA purification technique is basedupon use of organic solvents to extract contaminants from cell lysates.Thus, in one embodiment, the method comprises use of phenol, chloroformand isoamyl alcohol to extract the DNA. Suitable conditions are employedto ensure that the contaminants are separated into the organic phase andthat DNA remains in the aqueous phase.

In specific embodiments of these purification techniques, extracted DNAis recovered through alcohol precipitation, such as ethanol orisopropanol precipitation.

Amplification of DNA (using PCR) from natural sources is often inhibitedby co-purified contaminants and various methods adopted for DNAextraction from environmental samples are available and provide analternative for isolating DNA from faecal or blood based samples,according to specific embodiments of the invention. For instance, theQIAamp DNA Stool Mini Kit from QIAGEN adsorbs DNA-damaging substancesand PCR inhibitors present in the sample by InhibitEX. Other examplesfor application in particular to faecal samples include the WizardGenomic DNA Purification Kit (Promega), the NucliSENS® easyMAG™(Biomerieux) and nucleic acid purification kits manufactured by MachereyNagel.

In specific embodiments, where the test sample is a blood based sample,the DNA may be isolated by phenol-chloroform extraction since this hasbeen shown to provide particularly high levels of DNA recovery from thesample.

Where blood based test samples are employed, the ChargeSwitch proceduremay be utilised for example.

Suitable methods and kits for isolating DNA from blood samples arecommercially available. Examples, each of which may be utilised in themethods of the invention are provided in the table below.

TABLE 1 Kits and methods for isolating DNA from blood samples. KitCompany Method UltraClean-htp ™ BloodSpin ™ Mo Bio Silica-membrane DNALaboratories, Inc. PAXgene Blood DNA Kit Qiagen isopropanol QIAamp DNABlood Maxi/Mini Qiagen Silica-membrane Kit FlexiGene DNA Kit Qiagenisopropanol GeneCatcher gDNA 3-10 ml Invitrogen magnetic beads BloodBC-204-10 ml-blood - Blood 10 ml Baseclear magnetic beads ZR Genomic DNAI Kit Zymo research magnetic beads DNAzol BD MRC, Inc. isopropanolGentra pureGene* DNA Fischer isopropanol Purification Blood MasterPureWhole Blood DNA Epicentre isopropanol Biotech. Invisorb ® Blood Giga KitWestburg isopropanol 100436-10 (Maxi) Bioron Silica-membrane MagNA PureLC DNA Isolation Roche magnetic beads Kit Nuclisens EasyMag Biomérieuxmagnetic beads chemagic blood kit special chemagen magnetic beads

The QIAamp DNA Blood Maxi kit available from Qiagen and the GeneCatchergDNA kit from Invitrogen both utilise plasma or serum as startingmaterial.

Thus, as can be derived from table 1, DNA isolation may be carried outusing silica-membranes, isopropanol or magnetic bead based methods forexample.

The methods of the invention may also, as appropriate, incorporatequantification of isolated/extracted/purified DNA in the sample.Quantification of the DNA in the sample may be achieved using anysuitable means. Quantitation of nucleic acids may, for example, be basedupon use of a spectrophotometer, a fluorometer or a UV transilluminator.Examples of suitable techniques are described in standard texts such asMolecular Cloning—A Laboratory Manual (Third Edition), Sambrook andRussell (see in particular Appendix 8 therein). In one embodiment, kitssuch as the Picogreen dsDNA quantitation kit available from MolecularProbes, Invitrogen may be employed to quantify the DNA.

“Cancer” is defined herein to include neoplasias. Neoplasia refers toabnormal new growth and thus means the same as tumor, which may bebenign or malignant. Particular cancer types which are relevant inaccordance with the present invention are discussed above and includethose selected from neoplasias of the gastrointestinal tract. Specificexamples include colorectal cancer, oesophageal cancer, stomach cancerand gastric cancer.

“Colorectal cancer”, also called colon cancer or bowel cancer, isdefined to include cancerous growths in the colon, rectum and appendix.Specific markers and panels of markers, as described in greater detailherein, may be particularly applicable to certain cancer types.

Other cancer types which may be relevant in specific (but not all)embodiments of the invention include prostate cancer, breast cancer,ovarian cancer and thyroid cancer.

“Epigenetic silencing” is defined herein to include any alteration inthe DNA resulting in diminished gene expression which is mediated bymechanisms other than alterations in the primary nucleotide sequence ofa gene. Epigenetic modifications may, in certain circumstances be stableheritable traits. A number of related mechanisms that cause alterationin gene expression are recognised and include DNA methylation, histonechanges (for example changes in histone acetylation) which may lead tochromatin remodelling and RNA interference. In many cases,hypermethylation of DNA incorrectly switches off critical genes allowingcancers to develop and progress.

Epigenetic silencing of, or an epigenetic change such as methylation in,the at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) maymanifest itself before abnormal new growth/cancer is observable. Asubject may be undergoing routine screening and may not necessarily besuspected of having a disease such as a colon neoplasia. Detectingepigenetic silencing of the gene or genes in an adenoma of such asubject may indicate that the probable course of the adenoma isdevelopment to a carcinoma and thus there is a predisposition toneoplasia. In such cases, preventive treatment may be recommended andinvolve resection of the advanced adenoma.

These methods may advantageously involve detection of methylation of theNDRG4 gene, in particular using primer set 1, as discussed herein.

“Advanced adenoma” refers to an adenoma in which epigenetic silencing ofat least one of the gene linked to colorectal cancer is observed,preferably epigenetic silencing such as methylation of the gene or genesof the invention (such as NDRG4 etc.) is detected.

The most preferred epigenetic change in the at least one gene selectedfrom an NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR,GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations and combinationsincluding panels as discussed herein) which is detected comprises,consists essentially of or consists of methylation. In particular,aberrant methylation, which may be referred to as hypermethylation, ofthe gene or genes is detected. Typically, the methylation status isdetermined in suitable CpG islands which are often found in the promoterregion of the gene(s). The term “methylation”, “methylation state” or“methylation status” refers to the presence or absence of5-methylcytosine (“5-mCyt”) at one or a plurality of CpG dinucleotideswithin a DNA sequence. CpG dinucleotides are typically concentrated inthe promoter regions and exons of human genes.

Diminished gene expression can be assessed in terms of DNA methylationstatus or in terms of expression levels as determined by the methylationstatus of the gene. One method to detect epigenetic silencing is todetermine that a gene which is expressed in normal cells is lessexpressed or not expressed in tumor cells. Accordingly, the inventionprovides for a method of diagnosing cancer or predisposition to cancercomprising detecting epigenetic silencing of the NDRG4/NDRG2 subfamilygene, wherein epigenetic silencing of the NDRG2/NDRG4-family gene isdetermined by measurement of expression levels of the gene and whereinreduced expression of the gene is indicative for cancer orpredisposition to cancer. The invention also provides a method ofdetecting a predisposition to, or the incidence of, a cancer inparticular a gastrointestinal cancer such as colorectal cancer in asample comprising detecting an epigenetic change in at least one geneselected from GATA4, OSMR, NDRG4 (or another NDRG4/NDRG2 subfamilymember), GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC and, MGMT and/or atleast one gene selected from, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3and JAM3, wherein detection of the epigenetic change is determined bymeasuring expression levels of the at least one gene and wherein lowlevel, reduced level or a lack of expression of the at least one gene isindicative of a predisposition to, or the incidence of, cancer and inparticular a gastrointestinal cancer such as colorectal cancer.

In embodiments where blood and in particular plasma or serum samples areutilised, the at least one gene may be selected from OSMR, SFRP1, NDRG4,GATA5, ADAM23, JPH3, SFRP2 and APC and/or from TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3 and JAM3. These genes are also useful where faecaltest samples are employed. TFPI2 may be a particularly useful marker.Specific genes such as genes selected from TPF12, BNIP3, FOXE1, SYNE1and SOX17 may be most useful when plasma samples are employed. For stoolsamples genes such as GATA4, OSMR, NORG4, GATA5, SFRP1, ADAM23, JPH3,SFRP2, APC and MGMT and also genes selected from TFPI2, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 may usefully be employed. As shown below, thesegenes provide sensitive and specific methods for diagnosing colorectalcancer in plasma samples. Suitable panels in this context comprise,consist essentially of or consist of OSMR, NDRG4, GATA5 and ADAM23.Further panels are discussed below. This may be utilised in order todiagnose early stage colorectal cancer, in particular stage 0 to IIcolorectal cancer.

In embodiments in which tissue samples are utilised, the methodpreferably comprises, consists essentially of or consists of detectingan epigenetic change in a panel of genes comprising OSMR, GATA4 andADAM23 or OSMR, GATA4 and GATA5, wherein detection of the epigeneticchange in at least one of the genes in the panel is indicative of apredisposition to, or the incidence of, a gastrointestinal cancer suchas colorectal cancer. The tissue sample may comprise, consistessentially of or consist of a colon and/or rectal and/or appendixsample for example.

In specific embodiments, total loss of protein expression of the atleast one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) isobserved in the sample in order to conclude a diagnosis of cancer and inparticular a gastrointestinal cancer such as colorectal cancer orpredisposition to cancer and in particular a gastrointestinal cancersuch as colorectal cancer, or to make a decision on the best course oftreatment in accordance with the other methods of the invention, asdescribed herein (which description applies here mutatis mutandis).However, partial loss of expression of at least one gene selected froman NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) may also be relevant, due to methylation ofthe relevant gene or genes.

The decreased level of expression of at least one gene selected from anNDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) may, as necessary, be measured in order todetermine if it is statistically significant in the sample. This helpsto provide a reliable test for the methods of the invention. Any methodfor determining whether the expression level of at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) is significantlyreduced may be utilised. Such methods are well known in the art androutinely employed. For example, statistical analyses may be performed.One example involves an analysis of variance test. Typical P values foruse in such a method would be P values of <0.05 or 0.01 or 0.001 whendetermining whether the relative expression or activity is statisticallysignificant. A change in expression may be deemed significant if thereis at least a 10% decrease for example. The test may be made moreselective by making the change at least 15%, 20%, 25%, 30%, 35%, 40% or50%, for example, in order to be considered statistically significant.

In a specific embodiment of the methods of the invention, the decreasedlevel of expression or activity of the at least one gene selected froman NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) is determined with reference to a controlsample. This control sample is preferably taken from normal (i.e.non-tumorigenic) tissue in the subject, where expression of thecorresponding gene or genes is normal. Additionally, or alternatively,control samples may also be utilised in which there is known to be alack of expression of the at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein).

Suitable additional controls may also be included to ensure that thetest is working properly, such as measuring levels of expression oractivity of a suitable reference gene in both test and control samples.Suitable reference genes for the present invention include beta-actin,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA genessuch as 18S ribosomal RNA and RNA polymerase II gene (Radonic A. et al.,Biochem Biophys Res Commun. 2004 January 23; 313(4):856-62). In specificembodiments, the reference gene is beta-actin.

Expression of a nucleic acid can be measured at the RNA level or at theprotein level. Cells in test samples can be lysed and the mRNA levels inthe lysates, or in the RNA purified or semi-purified from the lysates,determined. Alternatively, methods can be used on unlysed tissues orcell suspensions. Suitable methods for determining expression of atleast one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) atthe RNA level are well known in the art and described herein.

Methods employing nucleic acid probe hybridization to the relevanttranscript(s) of the at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein) may be employed for measuring the presence and/or level of therespective mRNA. Such methods are well known in the art and include useof nucleic acid probe arrays (microarray technology) and Northern blots.Advances in genomic technologies now permit the simultaneous analysis ofthousands of genes, although many are based on the same concept ofspecific probe-target hybridization. Sequencing-based methods are analternative. These methods started with the use of expressed sequencetags (ESTs), and now include methods based on short tags, such as serialanalysis of gene expression (SAGE) and massively parallel signaturesequencing (MPSS). Differential display techniques provide yet anothermeans of analyzing gene expression; this family of techniques is basedon random amplification of cDNA fragments generated by restrictiondigestion, and bands that differ between two tissues identify cDNAs ofinterest.

In certain embodiments, the levels of gene expression of at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) are determined usingreverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is awell-known technique in the art which relies upon the enzyme reversetranscriptase to reverse transcribe mRNA to form cDNA, which can then beamplified in a standard PCR reaction. Protocols and kits for carryingout RT-PCR are extremely well known to those of skill in the art and arecommercially available.

In one embodiment, primers useful in RT-PCR carried out on the NDRG4gene are provided. These primers comprise, consist essentially of orconsist of the following sequences:

SEQ ID NO: 1 5′-cctgaggagaagccgctg-3′ (forward) SEQ ID NO: 25′-atgtcatgttccttccagtctgt-3′ (reverse) SEQ ID NO: 35′-GGCCTTCTGCATGTAGTGATCCG-3′ (forward) SEQ ID NO: 45′-GGTGATCTCCTGCATGTCCTCG-3′ (reverse)

Variants of these primers are included within the scope of theinvention, as defined herein which definition applies mutatis mutandis.

The RT-PCR can be carried out in a non-quantitative manner. End-pointRT-PCR measures changes in expression levels using three differentmethods: relative, competitive and comparative. These traditionalmethods are well known in the art. Alternatively, RT-PCR is carried outin a real time and/or in a quantitative manner. Real time quantitativeRT-PCR has been thoroughly described in the literature (see Gibson et alfor an early example of the technique) and a variety of techniques arepossible. Examples include use of hydrolytic probes (Taqman), hairpinprobes (Molecular Beacons), FRET probe pairs (LightCycler (Roche)),hairpin probes attached to primers (Scorpion), hairpin primers (Plexorand Amplifluor), DzyNA and oligonucleotide blocker systems. All of thesesystems are commercially available and well characterised, and may allowmultiplexing (that is, the determination of expression of multiple genesin a single sample).

TAQMAN was one of the earliest available real-time PCR techniques andrelies upon a probe which binds between the upstream and downstreamprimer binding sites in a PCR reaction. A TAQMAN probe contains a 5′fluorophore and a 3′ quencher moiety. Thus, when bound to its bindingsite on the DNA the probe does not fluoresce due to the presence of thequencher in close proximity to the fluorophore. During amplification,the 5′-3′ exonuclease activity of a suitable polymerase such as Taqdigests the probe if it is bound to the strand being amplified. Thisdigestion of the probe causes displacement of the fluorophore. Releaseof the fluorophore means that it is no longer in close proximity to thequencher moiety and this therefore allows the fluorophore to fluoresce.The resulting fluorescence may be measured and is in direct proportionto the amount of target sequence that is being amplified. These probesare sometimes generically referred to as hydrolytic probes.

In the Molecular Beacons system, the probe is again designed to bindbetween the primer binding sites. However, here the probe is a hairpinshaped probe. The hairpin in the probe when not bound to its targetsequence means that a fluorophore attached to one end of the probe and aquencher attached to the other end of the probe are brought into closeproximity and therefore internal quenching occurs. Only when the targetsequence for the probe is formed during the PCR amplification does theprobe unfold and bind to this sequence. The loop portion of the probeacts as the probe itself, while the stem is formed by complimentary armsequences (to respective ends of which are attached the fluorophore andquencher moiety). When the beacon probe detects its target, it undergoesa conformational change forcing the stem apart and this separates thefluorophore and quencher. This causes the energy transfer to thequencher to be disrupted and therefore restores fluorescence.

During the denaturation step, the Molecular Beacons assume a random-coilconfiguration and fluoresce. As the temperature is lowered to allowannealing of the primers, stem hybrids form rapidly, preventingfluorescence. However, at the annealing temperature, Molecular Beaconsalso bind to the amplicons, undergo conformational reorganisation,leading to fluorescence. When the temperature is raised to allow primerextension, the Molecular Beacons dissociate from their targets and donot interfere with polymerisation. A new hybridisation takes place inthe annealing step of every cycle, and the intensity of the resultingfluorescence indicates the amount of accumulated amplicon.

Scorpions primers are based upon the same principles as MolecularBeacons. However, here, the probe is bound to, and forms an integralpart of, an amplification primer. The probe has a blocking group at its5′ end to prevent amplification through the probe sequence. After oneround of amplification has been directed by this primer, the targetsequence for the probe is produced and to this the probe binds. Thus,the name “scorpion” arises from the fact that the probe as part of anamplification product internally hybridises to its target sequence thusforming a tail type structure. Probe-target binding is kineticallyfavoured over intrastrand secondary structures. Scorpions primers werefirst described in the paper “Detection of PCR products usingself-probing amplicons and fluorescence” (Nature Biotechnology. 17,p804-807 (1999)) and numerous variants on the basic theme havesubsequently been produced.

In similar fashion to Scorpions primers, Amplifluor primers rely uponincorporation of a Molecular Beacon type probe into a primer. Again, thehairpin structure of the probe forms part of an amplification primeritself. However, in contrast to Scorpions type primers, there is noblock at the 5′ end of the probe in order to prevent it being amplifiedand forming part of an amplification product. Accordingly, the primerbinds to a template strand and directs synthesis of the complementarystrand. The primer therefore becomes part of the amplification productin the first round of amplification. When the complimentary strand issynthesised amplification occurs through the hairpin structure. Thisseparates the fluorophore and quencher molecules, thus leading togeneration of florescence as amplification proceeds.

DzyNA primers incorporate the complementary/antisense sequence of a10-23 nucleotide DNAzyme. During amplification, amplicons are producedthat contain active (sense) copies of DNAzymes that cleave a reportersubstrate included in the reaction mixture. The accumulation ofamplicons during PCR/amplification can be monitored in real time bychanges in fluorescence produced by separation of fluorophore andquencher dye molecules incorporated into opposite sides of a DNAzymecleavage site within the reporter substrate. The DNAzyme and reportersubstrate sequences can be generic and hence can be adapted for use withprimer sets targeting various genes or transcripts (Todd et al.,Clinical Chemistry 46:5, 625-630 (2000)).

The Plexor™ qPCR and qRT-PCR Systems take advantage of the specificinteraction between two modified nucleotides to achieve quantitative PCRanalysis. One of the PCR primers contains a fluorescent label adjacentto an iso-dC residue at the 5′ terminus. The second PCR primer isunlabeled. The reaction mix includes deoxynucleotides and iso-dGTPmodified with the quencher dabcyl. Dabcyl-iso-dGTP is preferentiallyincorporated at the position complementary to the iso-dC residue. Theincorporation of the dabcyl-iso-dGTP at this position results inquenching of the fluorescent dye on the complementary strand and areduction in fluorescence, which allows quantitation duringamplification. For these multiplex reactions, a primer pair with adifferent fluorophore is used for each target sequence.

Real time quantitative techniques for use in the invention generallyproduce a fluorescent read-out that can be continuously monitored.Fluorescence signals are generated by dyes that are specific to doublestranded DNA, like SYBR Green, or by sequence-specificfluorescently-labeled oligonucleotide primers or probes. Each of theprimers or probes can be labelled with a different fluorophore to allowspecific detection. These real time quantitative techniques areadvantageous because they keep the reaction in a “single tube”. Thismeans there is no need for downstream analysis in order to obtainresults, leading to more rapidly obtained results. Furthermore, keepingthe reaction in a “single tube” environment reduces the risk of crosscontamination and allows a quantitative output from the methods of theinvention. This may be particularly important in a clinical setting forthe present invention.

It should be noted that whilst PCR is a preferred amplification method,to include variants on the basic technique such as nested PCR,equivalents may also be included within the scope of the invention.Examples include without limitation isothermal amplification techniquessuch as NASBA, 3 SR, TMA and triamplification, all of which are wellknown in the art and commercially available. Other suitableamplification methods without limitation include the ligase chainreaction (LCR) (Barringer et al, 1990), MLPA, selective amplification oftarget polynucleotide sequences (U.S. Pat. No. 6,410,276), consensussequence primed polymerase chain reaction (U.S. Pat. No. 4,437,975),invader technology (Third Wave Technologies, Madison, Wis.), stranddisplacement technology, arbitrarily primed polymerase chain reaction(WO90/06995) and nick displacement amplification (WO2004/067726).

Suitable methods for determining expression of at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) at the protein levelare also well known to one of skill in the art. Examples include westernblots, immunohistochemical staining and immunolocalization,immunofluorescence, enzyme-linked immunosorbent assay (ELISA),immunoprecipitation assays, complement fixation assay, agglutinationreactions, radioimmunoassay, flow cytometry, mass spectrophotometry, andequilibrium dialysis. These methods generally depend upon a reagentspecific for identification of the appropriate gene product. Anysuitable reagent may be utilised such as lectins, receptors, nucleicacids, antibodies etc. The reagent is preferably an antibody and maycomprise monoclonal or polyclonal antibodies. Fragments and derivatizedantibodies may also be utilised, to include without limitation Fabfragments, ScFv, single domain antibodies, nano-antibodies, heavy chainantibodies, aptamers etc. which retain gene product binding function.Any detection method may be employed in accordance with the invention.Proteins may be identified on the basis of charge, polarity, amino acidsequence etc. by a range of methods, including SDS-PAGE and amino acidsequencing for example. The nature of the reagent is not limited exceptthat it must be capable of specifically identifying the appropriate geneproduct.

Of course, in the case of a positive diagnosis of cancer and inparticular gastrointestinal cancer such as colorectal cancer, there willbe reduced levels of the relevant protein, and perhaps no protein atall. In one embodiment this will present a negative result, if theprotein specific reagent is one which binds to the wild type or fulllength protein. In this case, use of suitable controls ensures thatfalse diagnoses will not be made, for example caused by degraded ornon-specific reagents. Thus, the same reagent can be tested on samplesin which it is known that the at least one gene selected from anNDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) is expressed at the protein level. Apositive result in this control sample, combined with a negative resultin the test sample provides a confident diagnosis of cancer and removesany doubt over the quality of the reagent.

Measurement of expression of a gene on its own may not necessarilyconclusively indicate that the silencing is epigenetic, as the mechanismof silencing could be genetic, for example, by somatic mutation.Accordingly, in one embodiment, the methods of the invention incorporatean appropriate re-expression assay which is designed to reverseepigenetic silencing. Appropriate treatment of the sample using ademethylating agent, such as a DNA-methyltransferase (DMT) inhibitor mayreverse epigenetic silencing of the relevant gene. Suitable reagentsinclude, but are not limited to, DAC (5′-deazacytidine), TSA or anyother treatment affecting epigenetic mechanisms present in cell lines.Suitable reagents are discussed herein with respect to thepharmacogenetic and treatment aspects of invention, which discussionapplies mutatis mutandis. Typically, expression is reactivated orreversed upon treatment with such reagents, indicating that thesilencing is epigenetic.

As discussed in the experimental section, epigenetic silencing resultingin diminished expression of the NDRG4/NDRG2 subfamily gene has beenshown in a range of gastrointestinal cancers such as colorectal cancerand gastric cancer. Thus, in one embodiment, the invention provides fora method of diagnosing colorectal cancer and/or gastric cancer oranother gastrointestinal cancer as defined herein, predisposition tocolorectal cancer and/or gastric cancer or another gastrointestinalcancer as defined herein, comprising detecting epigenetic silencing ofthe NDRG4/NDRG2 subfamily gene, wherein epigenetic silencing of theNDRG2/NDRG4-family gene is determined by measurement of expressionlevels of the gene and wherein reduced expression of the gene isindicative for colorectal cancer and/or gastric cancer or anothergastrointestinal cancer as defined herein, predisposition to colorectalcancer and/or gastric cancer or another gastrointestinal cancer asdefined herein, or progression of adenoma to carcinoma. Preferably, thegene is NDRG2, or NDRG4, or a combination of NDRG2 and NDRG4.

As exemplified in the experimental section, epigenetic silencingresulting in diminished expression of the at least one gene selectedfrom GATA4, OSMR, NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT,TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 has been shown to besensitively and specifically linked with the incidence ofgastrointestinal cancer and in particular colorectal cancer. Thus, in afurther embodiment, the invention provides for a method of diagnosinggastrointestinal cancer and in particular colorectal cancer orpredisposition to gastrointestinal cancer and in particular colorectalcancer comprising detecting epigenetic silencing of at least one geneselected from GATA4, OSMR, NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2,APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3, whereinepigenetic silencing of the at least one gene selected from GATA4, OSMR,NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 is determined by measurement ofexpression levels of the gene and wherein reduced expression of the geneis indicative for gastrointestinal cancer and in particular colorectalcancer, predisposition to gastrointestinal cancer and in particularcolorectal cancer, or progression of adenoma to carcinoma. These markersmay usefully be employed when faecal test samples are utilised.

As is also discussed in the experimental section, epigenetic silencingresulting in diminished expression of the at least one gene selectedfrom GATA4, OSMR, NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC and MGMThas been shown to be sensitively and specifically linked with theincidence of colorectal cancer in specific tissue and bodily fluid, suchas faecal and blood-based samples. Thus, in one specific embodiment, theinvention provides for a method of diagnosing gastrointestinal cancerand in particular colorectal cancer or predisposition togastrointestinal cancer and in particular colorectal cancer comprisingdetecting epigenetic silencing of at least one gene selected from GATA4,OSMR, NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC and MGMT in atissue, faecal or a blood (plasma or serum) sample, or derivativethereof, wherein epigenetic silencing of the at least one gene selectedfrom GATA4, OSMR, NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC and MGMTis determined by measurement of expression levels of the gene andwherein reduced expression of the gene is indicative forgastrointestinal cancer and in particular colorectal cancer,predisposition to gastrointestinal cancer and in particular colorectalcancer, or progression of adenoma to carcinoma. As discussed above,where plasma or serum samples are utilised, the at least one gene may beselected from OSMR, SFRP1, NDRG4, GATA5, ADAM23, JPH3, SFRP2 and APC.

In alternative and complementary embodiments, in particular where bodilyfluid such as faecal and plasma samples are utilised the at least onegene may be selected from TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3. Panels may be selected from these genes and the other genes of theinvention as desired and as discussed herein. Methylation of these genesin stool and plasma samples has been shown for the first time herein tobe linked to colorectal cancer. Particularly useful markers, which givegood levels of sensitivity and specificity in both plasma and faecalsamples include TFPI2, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3. TFPI2 maybe particularly useful. Certain genes such as those selected from TFPI2,BNIP3, FOXE1, SYNE1 and SOX17 may prove most useful when testing plasmasamples. This discussion applies to all aspects of the invention asappropriate.

It is noted that the expression of additional genes may also bedetermined in order to supplement the methods of the invention. In fact,any gene involved in the establishment of cancer, as defined herein andin particular gastrointestinal cancers such as colorectal cancer,gastric cancer and/or oesophageal cancer, may be utilized in combinationwith the at least one gene selected from an NDRG2/NDRG4 subfamily gene(in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2,APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) inthe methods of present invention. In certain embodiments, the expressionlevel of the at least one gene selected from an NDRG2/NDRG4 subfamilygene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3,SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3(in all permutations and combinations including panels as discussedherein) is analysed in combination with at least one other gene involvedin the establishment of cancer. In one embodiment, at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) is combined with atleast two other genes involved in the establishment of cancer. In afurther embodiment at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein) and at least three, four, five or six other genes involved inthe establishment of cancer are combined. Other genes involved in theestablishment of (colorectal) cancer may be selected from the groupconsisting of CHFR, p16, Vimentin, p14, RASSF1a, RAB32, SEPTIN-9,RASSF2A, TMEFF2, NGFR and SMARCA3.

Since epigenetic silencing of the at least one gene selected from anNDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) manifests itself in methylation of the gene,the methods of the invention preferably involve detecting genemethylation. Accordingly, the invention provides a method of diagnosingcancer or predisposition to cancer, in particular gastrointestinalcancers such as colorectal cancer comprising detecting epigeneticsilencing of the at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein), wherein epigenetic silencing of the at least one gene isdetected by determination of the methylation status of the at least onegene and wherein methylation of the at least one gene is indicative forcancer or predisposition to cancer, as defined above and in particulargastrointestinal cancers such as colorectal cancer.

In embodiments where blood and in particular plasma or serum samples areutilised, the at least one gene may be selected from OSMR, SFRP1, NDRG4,GATA5, ADAM23, JPH3, SFRP2 and APC. As shown below, these genes providesensitive and specific methods for diagnosing colorectal cancer inplasma samples. Suitable panels in this context comprise, consistessentially of or consist of OSMR, NDRG4, GATA5 and ADAM23. This may beutilised in order to diagnose early stage colorectal cancer, inparticular stage 0 to II colorectal cancer. Additionally, oralternatively, the at least one gene may be selected from TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3, such as from TFPI2, BNIP3, FOXE1,SYNE1 and SOX17, in particular TFPI2.

In embodiments in which tissue samples are utilised, the methods maycomprise, consist essentially of or consist of detecting an epigeneticchange in a panel of genes comprising OSMR, GATA4 and ADAM23 or OSMR,GATA4 and GATA5, wherein detection of the epigenetic change in at leastone of the genes in the panel is indicative of a predisposition to, orthe incidence of, colorectal cancer. The tissue sample may comprise,consist essentially of or consist of a colon and/or rectal and/orappendix sample.

In embodiments where faecal samples are employed, the at least one genemay be selected from GATA4, OSMR, NDRG4, GATA5, SERP1, ADAM23, JPH3,SFRP2, APC and MGMT. In addition, or alternatively, the at least onegene may be selected from TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3,and JAM3, such as from TFPI2, FOXE1, SYNE1, SOZ17, PHACTR3 and JAM3, inparticular TFPI2. Two, three, four, five or six etc. gene panelsselected from these genes are also envisaged in the present invention.

CpG dinucleotides susceptible to methylation are typically concentratedin the promoter region, exons and introns of human genes. Promoter, exonand intron regions can be assessed for methylation. In one embodiment,the methylation status of the promoter region of the at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) is determined. A“promoter” is a region extending typically between approximately 1 Kb,500 bp or 150 to 300 bp upstream from the transcription start site.Frequently, the CpG island surrounding or positioned around thetranscription start site of the at least one gene selected from anNDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) is analysed to determine its methylationstatus. Alternatively, the methylation status of the exon and/or intronregions of the at least one gene selected from an NDRG2/NDRG4 subfamilygene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3,SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3(in all permutations and combinations including panels as discussedherein) may be determined as appropriate.

In one embodiment of the methods of the invention, the methylationstatus of the promoter region of the NDRG4 gene is analysed. In anotherembodiment, the methylation status of the promoter region of the NDRG2gene is analysed. Alternatively, the promoter region of NDRG2 and NDRG4are analysed simultaneously.

In one embodiment, the region of the NDRG4/NDRG2 subfamily genecomprising, consisting essentially of, or consisting of the nucleotidesequence of NDRG4 as set forth as SEQ ID NO: 524 (FIG. 3a ) and/or thenucleotide sequence of NDRG2 as set forth as SEQ ID NO: 525 (FIG. 3b )is analysed in order to determine its methylation status.

Various methylation assay procedures are known in the art, and can beused in conjunction with the present invention. These assays rely ontotwo distinct approaches: bisulphite conversion based approaches andnon-bisulphite based approaches. Non-bisulphite based methods foranalysis of DNA methylation rely on the inability ofmethylation-sensitive enzymes to cleave methylation cytosines in theirrestriction. The bisulphite conversion relies on treatment of DNAsamples with sodium bisulphite which converts unmethylated cytosine touracil, while methylated cytosines are maintained (Furuichi et al.,1970). This conversion results in a change in the sequence of theoriginal DNA.

DNA methylation analysis has been performed successfully with a numberof techniques including: sequencing, methylation-specific PCR (MS-PCR),melting curve methylation-specific PCR (McMS-PCR), MLPA with or withoutbisulfite treatment, QAMA (Zeschnigk et al, 2004), MSRE-PCR (Melnikov etal, 2005), MethyLight (Eads et al., 2000), ConLight-MSP (Rand et al.,2002), bisulfite conversion-specific methylation-specific PCR(BS-MSP)(Sasaki et al., 2003), COBRA (which relies upon use ofrestriction enzymes to reveal methylation dependent sequence differencesin PCR products of sodium bisulfite-treated DNA), methylation-sensitivesingle-nucleotide primer extension conformation (MS-SNuPE),methylation-sensitive single-strand conformation analysis (MS-SSCA),Melting curve combined bisulfite restriction analysis (McCOBRA)(Akey etal., 2002), PyroMethA, HeavyMethyl (Cottrell et al. 2004), MALDI-TOF,MassARRAY, Quantitative analysis of methylated alleles (QAMA), enzymaticregional methylation assay (ERMA), QBSUPT, MethylQuant, Quantitative PCRsequencing and oligonucleotide-based microarray systems, Pyrosequencing,Meth-DOP-PCR. A review of some useful techniques is provided in Nucleicacids research, 1998, Vol. 26, No. 10, 2255-2264, Nature Reviews, 2003,Vol. 3, 253 266; Oral Oncology, 2006, Vol. 42, 5-13, which referencesare incorporated herein in their entirety. Any of these techniques maybe utilised in accordance with the present invention, as appropriate.

Additional methods for the identification of methylated CpGdinucleotides utilize the ability of the methyl binding domain (MBD) ofthe MeCP2 protein to selectively bind to methylated DNA sequences (Crosset al, 1994; Shiraishi et al, 1999). Alternatively, the MBD may beobtained from MBP, MBP2, MBP4 or poly-MBD (Jorgensen et al., 2006). Inone method, restriction exonuclease digested genomic DNA is loaded ontoexpressed His-tagged methyl-CpG binding domain that is immobilized to asolid matrix and used for preparative column chromatography to isolatehighly methylated DNA sequences. Such methylated DNA enrichment-step maysupplement the methods of the invention. Several other methods fordetecting methylated CpG islands are well known in the art and includeamongst others methylated-CpG island recovery assay (MIRA). Any of thesemethods may be employed in the present invention where desired.

In specific embodiments, the methylation status of the at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) (or portion thereof,especially the CpG islands, as discussed herein) is determined usingmethylation specific PCR (MSP), or an equivalent amplificationtechnique. The MSP technique will be familiar to one of skill in theart. In the MSP approach, DNA may be amplified using primer pairsdesigned to distinguish methylated from unmethylated DNA by takingadvantage of sequence differences as a result of sodium-bisulphitetreatment (Herman et al., 1996; and WO 97/46705).

A specific example of the MSP technique is designated real-timequantitative MSP (QMSP), which permits reliable quantification ofmethylated DNA in real time. These methods are generally based on thecontinuous optical monitoring of an amplification procedure and utilisefluorescently labelled reagents whose incorporation in a product can bequantified and whose quantification is indicative of copy number of thatsequence in the template. One such reagent is a fluorescent dye, calledSYBR Green I that preferentially binds double-stranded DNA and whosefluorescence is greatly enhanced by binding of double-stranded DNA.Alternatively, labelled primers and/or labelled probes can be used. Theyrepresent a specific application of the well known and commerciallyavailable real-time amplification techniques such as hydrolytic probes(TAQMAN®), hairpin probes (MOLECULAR BEACONS®), hairpin primers(AMPLIFLUOR®), hairpin probes integrated into primers (SCORPION®),oligonucleotide blockers (such as the HeavyMethyl technique) and primersincorporating complementary sequences of DNAzymes (DzyNA®), specificinteratction between two modified nucleotides (Plexor™) etc as describedin more detail herein. Often, these real-time methods are used with thepolymerase chain reaction (PCR). In Heavymethyl, described for examplein WO02/072880 the priming is methylation specific, but non-extendableoligonucleotide blockers provide this specificity instead of the primersthemselves. The blockers bind to bisulfite-treated DNA in amethylation-specific manner, and their binding sites overlap the primerbinding sites. When the blocker is bound, the primer cannot bind andtherefore the amplicon is not generated. Heavymethyl can be used incombination with real-time or end point detection in the methods of theinvention.

Thus, in specific embodiments, the methylation status of the at leastone gene selected from an NDRG2/NDRG4 subfamily gene (in particularNDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT,TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutationsand combinations including panels as discussed herein) is determined bymethylation specific PCR/amplification, preferably real-time methylationspecific PCR/amplification. In specific embodiments, the real timePCR/amplification involves use of hairpin primers (Amplifluor)/hairpinprobes (Molecular Beacons)/hydrolytic probes (Taqman)/FRET probe pairs(Lightcycler)/primers incorporating a hairpin probe (Scorpion)/primersincorporating complementary sequences of DNAzymes that cleave a reportersubstrate included in the reaction mixture (DzyNA®)/fluorescent dyes(SYBR Green etc.)/oligonucleotide blockers/the specific interactionbetween two modified nucleotides (Plexor). Primers and/or probes can beused to investigate the methylation status of the at least one gene.

Real-Time PCR detects the accumulation of amplicon during the reaction.Real-time methods do not need to be utilised, however. Many applicationsdo not require quantification and Real-Time PCR is used principally as atool to obtain convenient results presentation and storage, and at thesame time to avoid post-PCR handling. Analyses can be performed only toknow if the target DNA is present in the sample or not. End pointverification is carried out after the amplification reaction hasfinished. This knowledge can be used in a medical diagnostic laboratoryto detect a predisposition to, or the incidence of, cancer in a patient.In the majority of such cases, the quantification of DNA template is notvery important. Amplification products may simply be run on a suitablegel, such as an agarose gel, to determine if the expected sized productsare present. This may involve use of ethidium bromide staining andvisualisation of the DNA bands under a UV illuminator for example.Alternatively, fluorescence or energy transfer can be measured todetermine the presence of the methylated DNA. The end-point PCRfluorescence detection technique can use the same approaches as widelyused for Real Time PCR: TaqMan assay, Molecular Beacons, Scorpion,Amplifluor etc. For example, «Gene» detector allows the measurement offluorescence directly in PCR tubes.

In real-time embodiments, quantitation may be on an absolute basis, ormay be relative to a constitutively methylated DNA standard, or may berelative to an unmethylated DNA standard. Methylation status may bedetermined by using the ratio between the signal of the marker underinvestigation and the signal of a reference gene where methylationstatus is known (such as β-actin for example), or by using the ratiobetween the methylated marker and the sum of the methylated and thenon-methylated marker. Alternatively, absolute copy number of themethylated marker gene can be determined. Suitable reference genes forthe present invention include beta-actin, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), ribosomal RNA genes such as 18S ribosomal RNA andRNA polymerase II gene (Radonic A. et al., Biochem Biophys Res Commun.2004 January 23; 313(4):856-62). In a particularly preferred embodiment,the reference gene is beta-actin.

In one embodiment, each clinical sample is measured in duplicate and forboth Ct values (cycles at which the amplification curves crossed thethreshold value, set automatically by the relevant software) copynumbers are calculated. The average of both copy numbers (for each gene)is used for the result classification. To quantify the final results foreach sample two standard curves are used, one for either the referencegene (β-actin or the non-methylated marker for example) and one for themethylated version of the marker. The results of all clinical samples(when m-Gene was detectable) are expressed as 1000 times the ratio of“copies m-Gene”/“copies reference gene” or “copies m-Gene”/“copiesu-Gene+m-Gene” and then classified accordingly (methylated,non-methylated or invalid) (u=unmethylated; m=methylated).

In one embodiment, primers useful in MSP carried out on the promoterregion of the NDRG4 gene are provided. These primers comprise, consistessentially of or consist of the following sequences:

TABLE 2 SEQ SEQ Number ID ID Annealing of PCR NO. NDRG4 PrimerSense primer NO. Antisense primer temp cycles  5 Primer FlankGGTTYGTTYGGGA  6 CRAACAACCAAA 56 35 set 1 TTAGTTTTAGG AACCCCTC  7 PrimerU GATTAGTTTTAGG  8 AAAACCAAACTA 66 25 set 1 TTTGGTATTGTTTT AAAACAATACACGT CA  9 Primer M TTTAGGTTCGGTA 10 CGAACTAAAAAC 66 25 set 1 TCGTTTCGCGATACGCCG 11 Primer Flank ATYGGGGTGTTTT 12 ATACCRAACCTA 56 35 set 2TTAGGTTT AAACTAATCCC 13 Primer U GGGTGTTTTTTAG 14 CCTAAAACTAATC 66 30set 2 GTTTCGCGTCGC CCAAACAAACCA 15 Primer M TTTTTTAGGTTTC 16AAACTAATCCCG 66 30 set 2 GCGTCGC AACGAACCG Where ″Flank″ = Flankingprimers ″U″ = Unmethylated NDRG4 specific primers ″M″ = Methylated NDRG4specific primers

Primer set 1 is useful in particular applications for predicting theprogression of adenomas.

Primer set 2 may provide slightly more sensitive results although bothprimer sets are clearly useful.

In a further embodiment, primers and probes useful in quantitative MSPcarried out on the (promoter region of the) NDRG4 gene are provided.These primers and probes comprise, consist essentially of or consist ofthe following sequences:

SEQ ID NO: 17 5′-GTATTTTAGTCGCGTAGAAGGC-3′ (forward primer)SEQ ID NO: 18 5′-AATTTAACGAATATAAACGCTCGAC-3′ (reverse primer) andSEQ ID NO: 19 5′-FAM-CGACATGCCCGAACGAACCGCGATCCCTGCATGTCG-3′-DABCYL (molecular beacon probe)

Further characteristics of these primers and probes are summarized inthe experimental part.

In a further embodiment, primers useful in MSP carried out on thepromoter region of the NDRG2 gene are provided. These primers comprise,consist essentially of or consist of the following sequences:

Flanking primers: SEQ ID NO: 20 5′-YGTTTTTTATTTATAGYGGTTTTT-3′(flank up) SEQ ID NO: 21 5′-TCCTAATACCTCTCCTCTCTTTACTAC-3′ (flank down)Unmethylated NDRG2 specific primers: SEQ ID NO: 225′-TTTTATTTATAGTGGTTTTTTGTATTTTTT-3′ (sense) SEQ ID NO: 235′-TCTCCTCTCTTTACTACATCCCAACA-3′(antisense)Methylated NDRG2 specific primers: SEQ ID NO: 245′-TTTATAGCGGTTTTTCGTATTTTTC-3′ (sense) SEQ ID NO: 255′-CCTCTCTTTACTACGTCCCGACG-3′ (antisense).

In one embodiment, primers and/or probes useful in determining themethylation status of the at least one gene selected from GATA4, OSMR,NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC and MGMT (carried out onthe promoter region of at least one gene selected from GATA4, OSMR,NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC and MGMT) are provided.These primers and/or probes comprise, consist essentially of or consistof the following sequences:

TABLE 3 Primer sequences and beacon (probe) sequences SEQ ID NO. 26β-Actin forward 5′-TAGGGAGTATATAGGTTGGGGAAGTT-3′ primer 27 reverse5′-AACACACAATAACAAACACAAATTCAC-3′ primer 28 beacon 5′-FAM-CGACTGCGTGTGGGGTGGTGATGGAGGAGGTTTAGGCAGTCG- 3′-DABCYL 29 GATA4 forward5′-AGGTTAGTTAGCGTTTTAGGGTC-3′ primer 30 reverse5′-ACGACGACGAAACCTCTCG-3′ primer 31 beacon 5′-FAM-CGACATGCCTCGCGACTCGAATCCCCGACCCAGCATGTCG-3′- DABCYL 32 GATA5 forward5′-AGTTCGTTTTTAGGTTAGTTTTCGGC-3′ primer 33 reverse5′-CCAATACAACTAAACGAACGAACCG-3 primer 34 beacon 5′-FAM-CGACATGCGTAGGGAGGTAGAGGGTTCGGGATTCGTAGCATGTCG- 3′-DABCYL 35 SFRP1forward 5′-TGTAGTTTTCGGAGTTAGTGTCGCGC-3′ primer 36 reverse5′-CCTACGATCGAAAACGACGCGAACG-3′ primer 37 beacon 5′-FAM-CGACATGCTCGGGAGTCGGGGCGTATTTAGTTCGTAGCGGCATGTCG- 3′-DABCYL 38 SFRP2forward 5′-GGGTCGGAGTTTTTCGGAGTTGCGC-3′ primer 39 reverse5′-CCGCTCTCTTCGCTAAATACGACTCG-3′ primer 40 beacon 5′-FAM-CGACATGCGGTGTTTCGTTTTTTCGCGTTTTAGTCGTCGGGCATGTCG- 3′-DABCYL 17 NDRG4forward 5′-GTATTTTAGTCGCGTAGAAGGC-3′ primer 18 reverse5′-AATTTAACGAATATAAACGCTCGAC-3′ primer 19 beacon5′-FAM-CGACATGCCCGAACGAACCGCGATCCCTGCATGTCG-3′- DABCYL 41 APC forward5′-GAACCAAAACGCTCCCCAT-3′ primer 42 reverse5′-TTATATGTCGGTTACGTGCGTTTATAT-3′ primer 43 beacon 5′-FAM-CGTCTGCCCCGTCGAAAACCCGCCGATTAACGCAGACG-3′- DABCYL 44 ADAM23 forward5′-GAAGGACGAGAAGTAGGCG-3′ primer 45 reverse5′-CTAACGAACTACAACCTTACCGA-3′ primer 46 beacon5′-FAM-CGACATGCCCCCGACCCGCACGCCGCCCTGCATGTCG- 3′-DABCYL 47 OSMR forward5′-TTTGGTCGGGGTAGGAGTAGC-3′ (3) primer 48 reverse5′-CGAACTTTACGAACGAACGAAC-3′ primer 49 beacon5′-FAM-CGACATGCCCGTACCCCGCGCGCAGCATGTCG-3′- DABCYL 47 OSMR forward5′-TTTGGTCGGGGTAGGAGTAGC-3′ (4) primer 50 reverse5′-AAAAACTTAAAAACCGAAAACTCG-3′ primer 49 beacon5′-FAM-CGACATGCCCGTACCCCGCGCGCAGCATGTCG-3′- DABCYL 51 JPH3 forward5′-TTAGATTTCGTAAACGGTGAAAAC-3′ primer

In specific embodiments, the methods of the invention employ or relyupon or utilise primers and/or probes selected from the primers andprobes comprising the nucleotide sequences set forth in Table 4 below todetermine the methylation status of the at least one gene. The tablepresents specific primer and probe combinations for certain preferredgenes whose methylation status may be determined according to themethods of the invention.

TABLE 4 Primer sequences and beacon (probe) sequences SEQ ID NO. 26β-Actin forward 5′-TAGGGAGTATATAGGTTGGGGAAGTT-3′ primer 27 reverse5′-AACACACAATAACAAACACAAATTCAC-3′ primer 28 beacon 5′-FAM-CGACTGCGTGTGGGGTGGTGATGGAGGAGGTTTAGGCAGTCG- 3′-DABCYL 29 GATA4 forward5′-AGGTTAGTTAGCGTTTTAGGGTC-3′ primer 30 reverse5′-ACGACGACGAAACCTCTCG-3′ primer 31 beacon 5′-FAM-CGACATGCCTCGCGACTCGAATCCCCGACCCAGCATGTCG-3′- DABCYL 32 GATA5 forward5′-AGTTCGTTTTTAGGTTAGTTTTCGGC-3′ primer 33 reverse5′-CCAATACAACTAAACGAACGAACCG-3 primer 34 beacon 5′-FAM-CGACATGCGTAGGGAGGTAGAGGGTTCGGGATTCGTAGCATGTCG- 3′-DABCYL 35 SFRP1forward 5′-TGTAGTTTTCGGAGTTAGTGTCGCGC-3′ primer 36 reverse5′-CCTACGATCGAAAACGACGCGAACG-3′ primer 37 beacon 5′-FAM-CGACATGCTCGGGAGTCGGGGCGTATTTAGTTCGTAGCGGCATGTCG- 3′-DABCYL 38 SFRP2forward 5′-GGGTCGGAGTTTTTCGGAGTTGCGC-3′ primer 39 reverse5′-CCGCTCTCTTCGCTAAATACGACTCG-3′ primer 40 beacon 5′-FAM-CGACATGCGGTGTTTCGTTTTTTCGCGTTTTAGTCGTCGGGCATGTCG- 3′-DABCYL 17 NDRG4forward 5′-GTATTTTAGTCGCGTAGAAGGC-3′ primer 18 reverse5′-AATTTAACGAATATAAACGCTCGAC-3′ primer 19 beacon5′-FAM-CGACATGCCCGAACGAACCGCGATCCCTGCATGTCG-3′- DABCYL 41 APC forward5′-GAACCAAAACGCTCCCCAT-3′ primer 42 reverse5′-TTATATGTCGGTTACGTGCGTTTATAT-3′ primer 43 beacon 5′-FAM-CGTCTGCCCCGTCGAAAACCCGCCGATTAACGCAGACG-3′- DABCYL 44 ADAM23 forward5′-GAAGGACGAGAAGTAGGCG-3′ primer 45 reverse5′-CTAACGAACTACAACCTTACCGA-3′ primer 46 beacon5′-FAM-CGACATGCCCCCGACCCGCACGCCGCCCTGCATGTCG- 3′-DABCYL 47 OSMR forward5′-TTTGGTCGGGGTAGGAGTAGC-3′ (3) primer 48 reverse5′-CGAACTTTACGAACGAACGAAC-3′ primer 49 beacon5′-FAM-CGACATGCCCGTACCCCGCGCGCAGCATGTCG-3′- DABCYL 47 OSMR forward5′-TTTGGTCGGGGTAGGAGTAGC-3′ (4) primer 50 reverse5′-AAAAACTTAAAAACCGAAAACTCG-3′ primer 49 beacon5′-FAM-CGACATGCCCGTACCCCGCGCGCAGCATGTCG-3′- DABCYL 51 JPH3 forward5′-TTAGATTTCGTAAACGGTGAAAAC-3 primer

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers and/or probes selected from the primerscomprising the nucleotide sequences set forth in Table 5 below todetermine the methylation status of NDRG4. The table presents specificprimer and probe combinations for determining the methylation status ofthis gene and the primer pairs and corresponding probe may be selectedaccording to the table.

TABLE 5Primer pairs and probes for determining the methylation status of NDRG4, withpredicted amplification product lengths shown. Ampli- SEQ Oligo Assaycon ID nucleotides 5′ to 3′ Sequences (all the name length NO & probesbeacons are 5′-FAM and 3′-DABCYL) NDRG4_1b 112 17 ForwardGTATTTTAGTCGCGTAGAAGGC primer 18 Reverse AATTTAACGAATATAAACGCTCGACprimer 57 Beacon CGACATGCAGGGATCGCGGTTCGTTCGGGCATGTCG NDRG4_13830 105 58Forward GGTATTTTAGTCGCGTAGAAGGC primer 59 Reverse GAATATAAACGCTCGACCCGCprimer 60 Beacon CGACATGCGCGGTTCGTTCGGGATTAGTTTTAGGTT CGGCATGTCGNDRG4_2(MvE)  88  9 Forward TTTAGGTTCGGTATCGTTTCGC primer 10 ReverseCGAACTAAAAACGATACGCCG primer 61 BeaconCGTACCCGCGTTTATATTCGTTAAATTTACGCGGGT ACG NDRG4_66292 163 62 ForwardTAGTCGCGTAGAAGGCGGA primer 63 Reverse GACTACAAAAACGAAAACCGAAC primer 64Beacon CGACATCGGGTACGTTTTCGCGGCGATGTCG NDRG4_66293 168 58 ForwardGGTATTTTAGTCGCGTAGAAGGC primer 65 Reverse CTACAAAAACGAAAACCGAAC primer66 Beacon CGTTTCGCGGGTCGAGCGAAACG NDRG4_66294 152 62 ForwardTAGTCGCGTAGAAGGCGGA primer 67 Reverse CGAAAACCGAACTAAAAACGA primer 68Beacon CGACATGCCGCGGTTCGTTCGGGATTAGTTTTAGGG CATGTCG NDRG4_66295  90 69Forward TTTCGTTCGTTTATCGGGT primer 70 Reverse CGAACCTAAAACTAATCCCGAACprimer 71 Beacon CGACACGCGTAGAAGGCGGAAGTTACGCGCGTGTCG NDRG4_66296 160 72Forward GGTTTCGTAGCGTATTTAGTATAGTTC primer 73 ReverseGTAACTTCCGCCTTCTACGC primer 74 BeaconCGACATGCGCGGATCGATCGGGGTGTTTTTTAGGGC ATGTCG NDRG4_66297 143 75 ForwardGAGTTGTTTTTGTCGTTTCGTTT primer 76 Reverse AACACCTTCATCTCGACGC primer 77Beacon CGACATGCGGTTCGGTCGAGCGCGCATGTCG NDRG4_66298 148 78 ForwardGTTGTGAGTTGTTTTTGTCGTTTC primer 76 Reverse AACACCTTCATCTCGACGC primer 79Beacon CGACATGCCGTTGTTTCGACGTCGTTATTTAGAGTC GGCATGTCG NDRG4_66299 144 80Forward TTTTAGTATTTTTATTTCGGCGTTC primer 81 Reverse CTACTCCTACCGCTTCGCTCprimer 82 Beacon CGACATCGCGCTCCTCTCCCCGATGTCG NDRG4_66300 151 83 ForwardCGGTGTTTTAGTATTTTTATTTCGG primer 84 Reverse AACTACTCCTACCGCTTCGCT primer85 Beacon CGACATCGGTTTTGGGTGGCGGCGATGTCG NDRG4_66301 120 80 ForwardTTTTAGTATTTTTATTTCGGCGTTC primer 86 Reverse CTCTCCTACCGCTCCGCTC primer87 Beacon CGACATCGCTCCTCTCCCCGACTCGATGTCG NDRG4_66302 125 83 ForwardCGGTGTTTTAGTATTTTTATTTCGG primer 86 Reverse CTCTCCTACCGCTCCGCTC primer88 Beacon CGACATGCCGAACGCGCTACCCCGCATGTCG NDRG4_66303  95 89 ForwardCGAGTCGTTTTAGTTTTCGGT primer 90 Reverse TACTCACAAATACCGCCCG primer 91Beacon CGACATCGGAAAGTGGCGGTCGGTTGCGATGTCG NDRG4_66304  85 92 ForwardTTCGGTGAATTTTAGGAGGC primer 93 Reverse TCGAACGACGAACACGAAA primer 94Beacon CGACATGCGCGGGGTGGGTGCGGCATGTCG

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers comprising thenucleotide sequences set forth in Table 6 and 7 below to determine themethylation status of GATA5. The table presents specific primercombinations for determining the methylation status of this gene and theprimer pairs may be selected according to the table. Table 6 also setsforth specific probes which may be utilised to facilitate (quantitative)detection of the methylation status of GATA5 and Table 7 incorporatesAmplifluour sequences which allow the primers to act as hairpin primers,thus facilitating quantitative detection (as discussed in detailherein).

TABLE 6Primer pairs and probes for determining the methylation status of GATA5, withpredicted amplification product lengths shown. Ampli- SEQ Oligo Assaycon ID nucleotides 5′ to 3′ Sequences (all the name length NO & probesbeacons are 5′-FAM and 3′-DABCYL) GATA5_12656  94  95 ForwardTTCGGGTTGGAGTATTTATTAGC primer  96 Reverse CGAACTTCCAATCTTCGACC primer 97 Beacon CGACATGCGGCGGTGGCGGTGGGTCGGCATGTCG GATA512659 102  98 ForwardGATTTTTCGGGGTTTACGAAG primer  99 Reverse GAAACTTAACGACAAAAACGCA primer100 Beacon CGACATGCGTTTAGTTGTATTGGTTCGGGTTTCGC ATGTCG GATA5_ 12666 107101 Forward GGTTTGTATTCGGATTCGGTC primer 102 ReverseTCGATAACAACGTCCTACACG primer 103 BeaconCGACATGCGAAGGTGGGTTTGCGGTTTGGGAGGTC GCATGTCG GATA5_ 12669 111 104Forward TAGGGTGCGGGTTTGTATTC primer 105 Reverse AACAACGTCCTACACGACCprimer 106 Beacon CGACATGCGTATTTATCGAAGGTGGGTTTGCGGTT TGCATGTCGGATA5_66212 118 107 Forward TAGTTGGTGTAGTAGAGGTCGGC primer 108 ReverseGACCTAAATCTCGCTTCCGT primer 109 BeaconCGACATGCCGAGGGAGATTGGAGTGAGTTTCGCAT GTCG GATA5_66213 139 110 ForwardTATAGCGTGGTGTTGGTCGT primer 111 Reverse CTAAATCTCGCTTCCGTCC primer 112Beacon CGACATGCGCGAGGGAGATTGGAGTGAGTTTCGCA TGTCG GATA5_66215  80 113Forward GGTGTCGAGGTTTTTAAGGTTTC primer 114 ReverseTCACTTTCTAACGAAAACGACT primer 115 BeaconCGACATGCGGGACGGGATGGGTTTTTGCGGGCATG TCG GATA5_66216 124 116 ForwardGTAGTTTCGGAGTTGGGTGTC primer 117 Reverse AAAAACGACTCTTCCCGATT primer 118Beacon CGACATGCGAGGGACGGGATGGGTTTTTGCATGTC G GATA5_66217 118 116 ForwardGTAGTTTCGGAGTTGGGTGTC primer 119 Reverse GACTCTTCCCGATTACAACG primer 120Beacon CGACATGCGAGGGACGGGATGGGTTTTTGGCATGT CG GATA5_66218  71 121Forward TTTTGCGTTAAAGGGTCGG primer 122 Reverse CGAAACCTTAAAAACCTCGACAprimer 123 Beacon CGACATGCCGGGGTTTTAAAGGTAGTTTCGGAGTT GGCATGTCGGATA5_66219  90 124 Forward GATGTCGTTGCGTTCGTTT primer 125 ReverseCCGAAACCTTAAAAACCTCG primer 126 BeaconCGACATGCCGGCGGGGTTTTAAAGGTAGTTTCGGC ATGTCG GATA5_66220  98 127 ForwardGTTTTGCGGATGTCGTTGC primer 125 Reverse CCGAAACCTTAAAAACCTCG primer 126Beacon CGACATGCCGGCGGGGTTTTAAAGGTAGTTTCGGC ATGTCG GATA5_66221 158 128Forward TAGGGGTTTTGCGGATGTC primer 114 Reverse TCACTTTCTAACGAAAACGACTprimer 126 Beacon CGACATGCCGGCGGGGTTTTAAAGGTAGTTTCGGC ATGTCG GATA5_66222150 129 Forward TCGAGATTGTGGAGTTTTCGT primer 130 ReverseTAAAAACCTCGTACTCCGCC primer 131 BeaconCGACATCGGTTTGGGAGGTCGTGTAGGACGATGTC G GATA5_66223 103 129 ForwardTCGAGATTGTGGAGTTTTCGT primer 132 Reverse GTAACCCAATCCTAAACTACCGA primer131 Beacon CGACATCGGTTTGGGAGGTCGTGTAGGACGATGTC G GATA5_66224 112 133Forward GGTTTGTATTCGGATTCGGT primer 134 Reverse ACCCTTCGATAACAACGTCCprimer 135 Beacon CGACATGCCGTATTTATCGAAGGTGGGTTTGCGGG CATGTCGGATA5_66225  76 136 Forward GTTTCGAGATTGTGGAGTTTTC primer 137 ReverseGATAACAACGTCCTACACGACC primer 138 BeaconCGACATGCCGAAGGTGGGTTTGCGGTTTGGGGCAT GTCG GATA5_66226 163 139 ForwardTTATTCGTTTCGTTTCGGG primer 140 Reverse AAACCCACCTTCGATAAATACG primer 141Beacon CGACATCGTTTTTGGTAGGGAGGTTCGGATCGATG TCG GATA5_66227 164 142Forward CGGGGTGTTATTTAGGTTTATTC primer 143 ReverseAATACGAAAACTCCACAATCTCG primer 144 BeaconCGACATGCGTTTTTGGTAGGGAGGTTCGGATCGCA TGTCG GATA5_66228  76 145 ForwardCGTTTTTGGTAGGGAGGTTC primer 146 Reverse ATCCGAATACAAACCCGCA primer 147Beacon CGACATGCCGTGGGGGAGGATGAGGGGAGCGTTTC GGCATGTCG GATA5_66229 113 142Forward CGGGGTGTTATTTAGGTTTATTC primer 148 Reverse AAACCCGCACCCTACGAAAprimer 144 Beacon CGACATGCGTTTTTGGTAGGGAGGTTCGGATCGCA TGTCG GATA5_66230161 149 Forward ATTAGTGTAGTTAGACGGGCGG primer 150 ReverseGACTCAACCACCAAACACGA primer 151 Beacon CGACATGCGTGGGTTTCGGGGAGTCGCATGTCGGATA5_66231 116  95 Forward TTCGGGTTGGAGTATTTATTAGC primer 152 ReverseAAACTACGAAACCTCAACGACC primer 153 Beacon CGACATGCGGTGGCGGTGGGTCGCATGTCGGATA5_66233 134 154 Forward GTTACGGGAGTTTTGCGTTT primer 155 ReverseCGATTCCTCTCCCTCGAAT primer 156 BeaconCGACATGCGAGTTTATGTCGGGTAGGTGTCGCATG TCG GATA5_66234 105 157 ForwardAATCGTGTTTCGTTCGTATTTTC primer 158 Reverse GATATACTCCGAACCCGCC primer159 Beacon CGACATGCGCGGAGTAGTTTCGTAGGTTGCGGGCA TGTCG GATA5_66235 121 160Forward GCGATTTAGGTTAGGGAATCGT primer 158 Reverse GATATACTCCGAACCCGCCprimer 161 Beacon CGACATGCCGGTGAGGGTTGTATGGAGGCGTCGGC ATGTCG GATA5_66237 99 162 Forward TTTCGGTGGGGTTTTTAGTC primer 163 ReverseGATTCCCTAACCTAAATCGCCT primer 164 BeaconCGACATGCGCGTTAGAAATGCGTGTGGGTAGGAGG CGCATGTCG GATA5_66238  72 165Forward ATTTCGGTGGGGTTTTTAGTC primer 166 Reverse CACACGCATTTCTAACGCCprimer 167 Beacon CGACATGCCTCTTCCCGAATCCCCGAAAACCGCAT GTCG GATA5_66243 91 168 Forward GGGTTTTATCGTCGCGTGT primer 169 ReverseCCGAAAACTAACCTAAAAACGAA primer 170 BeaconCGACATGCCCCGACCCCGCTCACCGGCATGTCG GATA5_66244 100 171 ForwardGGGGTTTACGGGGTTTTATC primer 172 Reverse CGAAAACTAACCTAAAAACGAAC primer173 Beacon CGACATGCGATAATCCCGACCCCGCTCACCGCATG TCG GATA5_66245 152 174Forward TTGTTTAGAAATCGAGGAAATCG primer 175 Reverse CGACGATAAAACCCCGTAAprimer 176 Beacon CGACATGCGAGTTTCGGGTGCGGTTACGCATGTCG GATA5_66247 163177 Forward TGTGGTTTCGTTTGTTTAGAAATC primer 175 ReverseCGACGATAAAACCCCGTAA primer 178 BeaconCGACATGCGAGTTTCGGGTGCGGTTACGTAACGCA TGTCG GATA5_66250 151 177 ForwardTGTGGTTTCGTTTGTTTAGAAATC primer 179 Reverse CCCGTAAACCCCCTCGTTA primer180 Beacon CGACATGCCGCGGGGTTTTCGTTAGTGTATTTCGG CATGTCG GATA5_66251  85181 Forward CGTTTGTTTAGAAATCGAGGAAATC primer 182 ReverseCATAAAAACGACCGACTCGAA primer 183 BeaconCGACATGCGGGGTTTTCGTTAGTGTATTTCGTTTT AGCATGTCG GATA5_66252 141 184Forward TTCGTATTTCGTTATTTATTCGGTT primer 185 ReverseGAAACTATAAAACCCCCGCA primer 186 BeaconCGACATGCCGGGTTTTTCGATGGTAGCGTTTTGTA CGGCATGTCG GATA5_66254 131 187Forward CGAGTTTTCGTTAGGTCGTTT primer 188 Reverse ACTCGACTCACACCCGAACprimer 189 Beacon CGACATGCGTACGTTTCGGGCGTCGGTTTTTCGGC ATGTCG GATA5_66255119 190 Forward CGCGAGTTTTCGTTAGGTC primer 191 ReverseCGAACAAATAAAACAACATCGAA primer 189 BeaconCGACATGCGTACGTTTCGGGCGTCGGTTTTTCGGC ATGTCG GATA5_66256  95 192 ForwardTCGGGATTTTGGAGGTTTC primer 193 Reverse CTACGAATACCGCTACGCC primer 194Beacon CGACATGCGGGATTTCGTCGGTTTTTTGGCGTAGG GCATGTCG

TABLE 7Additional assay designs: Primer and amplifluor sequences for determining themethylation status of GATA5, with predicted amplification product lengths shown.Ampli- SEQ Assay con ID Oligo name length NO nucleotides 5′ to 3′Sequences GATA5_12671_  90 195 Forward AGCGATGCGTTCGAGCATCGCUTTTTTCGAS_AMP primer TGTTGTTTTATTTGTTC 196 Reverse ATAACTATCTACGCCCAACCGA primerGATA5_12671_  90 197 Forward TTTTTCGATGTTGTTTTATTTGTTC AS_AMP primer 198Reverse AGCGATGCGTTCGAGCATCGCUATAACTAT primer CTACGCCCAACCGAGATA5_66214_  70 199 Forward AGCGATGCGTTCGAGCATCGCUTTCGTGTA S_AMP primerGTTTTATGTAGAGGTCG 200 Reverse GCTATAACGACGAAACTCGAA primer GATA5_66214_ 70 201 Forward TTCGTGTAGTTTTATGTAGAGGTCG AS_AMP primer 202 ReverseAGCGATGCGTTCGAGCATCGCUGCTATAAC primer GACGAAACTCGAA GATA5_66236_  73 203Forward AGCGATGCGTTCGAGCATCGCUTTAGGCGT S_AMP primer TAGAAATGCGTG 204Reverse CACCGAAAATACGAACGAAA primer GATA5_66236_  73 205 ForwardTTAGGCGTTAGAAATGCGTG AS_AMP primer 206 ReverseAGCGATGCGTTCGAGCATCGCUCACCGAAA primer ATACGAACGAAA GATA5_66239_ 101 207Forward AGCGATGCGTTCGAGCATCGCUGGTCGTTA S_AMP primer AGTTTGGGTTTATTC 208Reverse AAAACTACATAAAAACGCCGCTA primer GATA5_66239_ 101 209 ForwardGGTCGTTAAGTTTGGGTTTATTC AS_AMP primer 210 ReverseAGCGATGCGTTCGAGCATCGCUAAAACTAC primer ATAAAAACGCCGCTA GATA5_66240_  93207 Forward AGCGATGCGTTCGAGCATCGCUGGTCGTTA S_AMP primer AGTTTGGGTTTATTC211 Reverse ATAAAAACGCCGCTACCGC primer GATA5_66240_  93 209 ForwardGGTCGTTAAGTTTGGGTTTATTC AS_AMP primer 212 ReverseAGCGATGCGTTCGAGCATCGCUATAAAAAC primer GCCGCTACCGC GATA5_66241_  78 213Forward AGCGATGCGTTCGAGCATCGCUCGTTAAGT S_AMP primer TTGGGTTTATTCGGT 214Reverse CTACCGCGAAACAACTCCG primer GATA5_66241_  78 215 ForwardCGTTAAGTTTGGGTTTATTCGGT AS_AMP primer 216 ReverseAGCGATGCGTTCGAGCATCGCUCTACCGCG primer AAACAACTCCG GATA5_66248_  86 217Forward AGCGATGCGTTCGAGCATCGCUGTTTAGAA S_AMP primer ATCGAGGAAATCGC 218Reverse GACTTCCATAAAAACGACCGA primer GATA5_66248_  86 219 ForwardGTTTAGAAATCGAGGAAATCGC AS_AMP primer 220 ReverseAGCGATGCGTTCGAGCATCGCUGACTTCCA primer TAAAAACGACCGA GATA5_66249_  80 217Forward AGCGATGCGTTCGAGCATCGCUGTTTAGAA S_AMP primer ATCGAGGAAATCGC 182Reverse CATAAAAACGACCGACTCGAA primer GATA5_66249_  80 219 ForwardGTTTAGAAATCGAGGAAATCGC AS_AMP primer 221 ReverseAGCGATGCGTTCGAGCATCGCUCATAAAAA primer CGACCGACTCGAA GATA5_66257_  78 222Forward AGCGATGCGTTCGAGCATCGCUTTTGCGTG S_AMP primer GTCGTAAGGTC 223Reverse AAATAAACCCCGAACCGAA primer GATA5_66257_  78 224 ForwardTTTGCGTGGTCGTAAGGTC AS_AMP primer 225 ReverseAGCGATGCGTTCGAGCATCGCUAAATAAAC primer CCCGAACCGAA GATA5_66246_  70 226Forward AGCGATGCGTTCGAGCATCGCUCGGGGTTT S_AMP primer TCGTTAGTGTATTTC 227Reverse AAACCGACTTCCATAAAAACGA primer GATA5_66246_  70 228 ForwardCGGGGTTTTCGTTAGTGTATTTC AS_AMP primer 229 ReverseAGCGATGCGTTCGAGCATCGCUAAACCGAC primer TTCCATAAAAACGA

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers comprising thenucleotide sequences set forth in Tables 8 and 9 below to determine themethylation status of OSMR. The tables present specific primercombinations for determining the methylation status of this gene and theprimer pairs may be selected according to the table. Table 8 also setsforth specific probes which may be utilised to facilitate (quantitative)detection of the methylation status of OSMR and Table 9 incorporatesAmplifluour sequences which allow the primers to act as hairpin primers,thus facilitating quantitative detection (as discussed in detailherein).

TABLE 8Primer pairs and probes (molecular beacons) for determining the methylation statusof OSMR, with predicted amplification product lengths shown. Ampli- SEQOligo Assay con ID nucleotides 5′ to 3′ Sequences (all the name lengthNO & probes beacons are 5′-FAM and 3′-DABCYL) OSMR_1 148 230 ForwardGTGTTAAGAGTGCGTAGTAAGACG primer 231 Reverse GAAACGAACGTACAAAAACGA primer232 Beacon CGACATGCCGAAACTATAAATCAACTAC GAAACAAACGCGCATGTCG OSMR_2 142233 Forward TTAAGTAAACGTTGGGTAGAGGC Primer 234 ReverseCTCGATAACTTTTCCGACGA primer 235 Beacon CGACATGCCGAGGAGGGGAACGGGTTGTTGGCATGTCG OSMR_25259 138 236 Forward TGTTCGTTCGTTCGTAAAGTTC primer 237Reverse TACAATTTCCCGTCTTACTACGC primer 238 BeaconCGACATGCGCGGTCGTTTTTTTTCGGGA TTGAAGGCATGTCG OSMR_25260 139  47 ForwardTTTGGTCGGGGTAGGAGTAGC primer 239 Reverse CACAACCCGAACTTTACGAAC primer240 Beacon CGACATGCGCGGGGTACGGAGTTTCGGT CGCATGTCG OSMR_5 130 241 ForwardACGTTGGGTAGAGGCGGTATC primer 242 Reverse ATAACTTTTCCGACGAACGAAC primer243 Beacon CGACATGCACCCATCCCGACTAAACGCG ACGCATGTCG OSMR_66307 120 244Forward GTATAGTACGGGGTTCGTTCGT primer 245 Reverse ACTCGTAAAACCCTTCGCCprimer 246 Beacon CGACATGCGGTAGGGCGCGAGTAGAGCG CATGTCG OSMR_66308 124247 Forward GGTAGAGGCGGTATCGAGG primer 242 ReverseATAACTTTTCCGACGAACGAAC primer 248 Beacon CGACATGCGGGATGGGTTGCGAAGTTGTCGCATGTCG OSMR_66309 130 249 Forward ACGTTGGGTAGAGGCGGTA primer 242Reverse ATAACTTTTCCGACGAACGAAC primer 250 BeaconCGACACGCGTTTAGTCGGGATGGGTTGC GTGTCG OSMR_66310  76 251 ForwardCGGTATCGAGGAGGGGAAC primer 252 Reverse AAATCCGACAACTTCGCAA primer 253Beacon CGACATGCGTTGTTGTATTTTCGGTCGC GTTTAGTCGCATGTCG OSMR_66311  84 247Forward GGTAGAGGCGGTATCGAGG primer 252 Reverse AAATCCGACAACTTCGCAAprimer 254 Beacon CGACATGCCGGGTTGTTGTATTTTCGGT CGCGGCATGTCG OSMR_66312120 255 Forward TAGGTAGGTAGGTCGGGGGC primer 256 ReverseCGAAAATACAACAACCCGTTC primer 257 Beacon CGACATGCGTTGGGTAGAGGCGGTATCGCATGTCG OSMR_Sid 142 258 Forward TTCGTGCGTTTTTGGTCG primer 259 ReverseCGAACTTTACGAACGAACG primer 240 Beacon CGACATGCGCGGGGTACGGAGTTTCGGTCGCATGTCG

TABLE 9Additional assay designs: Primer and amplifluor sequences for determining themethylation status of OSMR, with predicted amplification product lengths shown.Ampli- SEQ Assay con ID Oligo name length NO nucleotides 5′ to 3′Sequences OSMR_25258_ 135 260 Forward AGCGATGCGTTCGAGCATCGCUAGAG S_AMPprimer TGCGTAGTAAGACGGGA 261 Reverse ACGTACAAAAACGACCCGAAC primerOSMR_25258_ 135 262 Forward AGAGTGCGTAGTAAGACGGGA AS_AMP primer 263Reverse AGCGATGCGTTCGAGCATCGCUACGT primer ACAAAAACGACCCGAAC OSMR_25264_ 65 264 Forward AGCGATGCGTTCGAGCATCGCUGCGT S_AMP primerAGCGTTGTTTTTGTTTC 265 Reverse CGACTTACCTCTAATTCCGCC primer OSMR_25264_ 65 266 Forward GCGTAGCGTTGTTTTTGTTTC AS_AMP primer 267 ReverseAGCGATGCGTTCGAGCATCGCUCGAC primer TTACCTCTAATTCCGCC OSMR_66305_ 142 260Forward AGCGATGCGTTCGAGCATCGCUAGAG S_AMP primer TGCGTAGTAAGACGGGA 231Reverse GAAACGAACGTACAAAAACGA primer OSMR_66305_ 142 262 ForwardAGAGTGCGTAGTAAGACGGGA AS_AMP primer 268 ReverseAGCGATGCGTTCGAGCATCGCUGAAA primer CGAACGTACAAAAACGA OSMR_66306_  98 260Forward AGCGATGCGTTCGAGCATCGCUAGAG S_AMP primer TGCGTAGTAAGACGGGA 269Reverse CTACGAAACAAACGCGAAA primer OSMR_66306_  98 262 ForwardAGAGTGCGTAGTAAGACGGGA AS_AMP primer 270 ReverseAGCGATGCGTTCGAGCATCGCUCTAC primer GAAACAAACGCGAAA OSMR_66313_  71 271Forward AGCGATGCGTTCGAGCATCGCUCGAG S_AMP primer GATTTTTCGAGCGTC 272Reverse ATACCGCCTCTACCCAACG primer OSMR_66313_  71 273 ForwardCGAGGATTTTTCGAGCGTC AS_AMP primer 274 Reverse AGCGATGCGTTCGAGCATCGCUATACprimer CGCCTCTACCCAACG

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers comprising thenucleotide sequences set forth in Table 10 below to determine themethylation status of ADAM23. The table presents specific primercombinations for determining the methylation status of this gene and theprimer pairs may be selected according to the table. Table 6 also setsforth specific probes which may be utilised to facilitate (quantitative)detection of the methylation status of ADAM23.

TABLE 10Primer pairs and probes (molecular beacons) for determining the methylationstatus of ADAM23, with predicted amplification product lengths shown.Ampli- SEQ Assay con ID Oligo 5′ to 3′ Sequences (all the name length NOnucleotides beacons are 5′-FAM and 3′-DABCYL) ADAM23_5  99 275 ForwardTAACGTAAAGGGTACGGGG primer 276 Reverse GTCCTTCTCCTACTACCTCCGCT primer277 Beacon CGACATGCCCCGACTCGCCTAACCTC GCAAGCATGTCG ADAM23_66258  98 278Forward GTAGTAGTTCGCGGTAGTCGTTT primer 279 Reverse AACGCTAACAAACACCGAAprimer 280 Beacon CGACATGCGCGGGTTGTAGTTTTGTC GGCGGCATGTCG ADAM23_66259169 281 Forward TTCGTAGTCGTTGAAGCGG primer 282 ReverseGCGAAACTCGAAACTAAACGA primer 283 Beacon CGACATCGGGAGTGGTTGCGAGGTTAGGCGATGTCG ADAM23_66260  81 284 Forward GCGTCGTTTTAGTATTTTTAGGTTC primer285 Reverse GACTACTCCCTCCCCCGAC primer 286 BeaconCGACATGCGTTTTCGTAGTCGTTGAA GCGGTCGGCATGTCG ADAM23_66261 104 287 ForwardGTTTTCGCGTCGTTCGTTT primer 285 Reverse GACTACTCCCTCCCCCGAC primer 288Beacon CGACATGCGGTTCGGCGGTAGTTTTC GTAGTCGGCATGTCG ADAM23_66263 106 289Forward GGGTACGGGGTTATATTTATCGT primer 290 Reverse CTACCGCCTACTTCTCGTCCPrimer 291 Beacon CGACATCGGGACGAGGCGGCGATGTC G ADAM23_66264  90 289Forward GGGTACGGGGTTATATTTATCGT primer 276 ReverseGTCCTTCTCCTACTACCTCCGCT primer 292 Beacon CGACATGCCCCCGCGCCTAAAAAACTACTACGGCATGTCG ADAM23_66265  84 293 Forward GGTACGGGGTTATATTTATCGTTGprimer 294 Reverse TCTCCTACTACCTCCGCTCG primer 295 BeaconCGACATGCCTCGTCCCGACCCCGCGC ATGTCG ADAM23_66266 125 296 ForwardGTCGAGTCGGGGATAAGTTC primer 297 Reverse AAAAACTACTACGCCCAACGA primer 298Beacon CGACATGCGCGGGAAAGTTAACGTAA AGGGTACGCATGTCG ADAM23_66267  97 296Forward GTCGAGTCGGGGATAAGTTC primer 299 Reverse AACCCCGTACCCTTTACGTTprimer 300 Beacon CGACGCGCGTTTTTCGTTTTTTTTTG TAGGGTTTCGCGTCGADAM23_66268 133 301 Forward AAGGAAAGGTCGAGTCGGG primer 297 ReverseAAAAACTACTACGCCCAACGA primer 302 Beacon CGACATGCGTAGGGTTTCGCGGGAAAGTTAACGGCATGTCG ADAM23_66269 108 301 Forward AAGGAAAGGTCGAGTCGGG primer303 Reverse TATAACCCCGTACCCTTTACGTT primer 304 BeaconCGACATGCAGTTCGGAGTATACGGAT TCGCGCGCATGTCG ADAM23_66271  97 305 ForwardTTCGTCGGTTATACGGAGC primer 306 Reverse GACAAAACTACAACCCGCCA primer 307Beacon CGACATGCGGGAGTTATGAGTTATGA AGTCGTTCGCATGTCG ADAM23_A 112 308Forward GAGGTTTTAAGTTGGCGGAGC primer 309 Reverse ACTCGAAACTAAACGACGCCCprimer 277 Beacon CGACATGCCCCGACTCGCCTAACCTC GCAAGCATGTCG

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers comprising thenucleotide sequences set forth in Table 11 below to determine themethylation status of JPH3. The table presents specific primercombinations for determining the methylation status of this gene and theprimer pairs may be selected according to the table. Table 11 also setsforth specific probes which may be utilised to facilitate (quantitative)detection of the methylation status of JPH3.

TABLE 11Primer pairs and probes (molecular beacons) for determining the methylationstatus of JPH3, with predicted amplification product lengths shown.Ampli- SEQ 5′ to 3′ Sequences (all con ID Oligothe beacons are 5′-FAM and Assay name length NO nucleotides 3′-DABCYL)JPH3_1(MVE) 103 310 Forward TTTAATATGGTGTAGTCGTTAGCGTC primer 311Reverse CCCACCTACGACTACCGCG primer 312 Beacon CGACATGCACGAAACCCGCGAACGACGACGCATGTCG JPH3_12608  90 313 Forward GGGGTAGGTTTAATTTTGACGAC primer314 Reverse TAAAACCGATACAAACGCCA primer 315 BeaconCGACATGCGGTTGGGAGGACGGTAAG GCGGCATGTCG JPH3_2 123 316 ForwardTGTAGTCGTTAGCGTCGTCGT primer 317 Reverse GAAAAACAACTCAAACCCGAA primer318 Beacon CGACATGCACCCGCGAACGACGACGA CGCATGTCG JPH3_3  88 319 ForwardGTAGGTTTAATTTTGACGACGGA primer 320 Reverse TTAAAACCGATACAAACGCCA primer321 Beacon CGACATGCCCGTACGCCTTACCGTCC TCGCATGTCG JPH3_4 134 322 ForwardGATATAGTAGAGTCGCGGTCGTC primer 323 Reverse CGATTAACTAAAATTCCTCCGAAAprimer 324 Beacon CGACATGCCCGAAAAACGCTCGCGAC CCAGCATGTCG JPH3_5 127 325Forward GGGGTAGTTTAGGTTCGGGTC primer 326 Reverse ATATAATACAACCGCCAACGCCprimer 327 Beacon CGACATGCCCGCAACGCGACAACCGC AGCATGTCG JPH3_67326 122328 Forward GTAGTCGTTAGCGTCGTCGT primer 317 ReverseGAAAAACAACTCAAACCCGAA primer 329 Beacon CGACATGCGCGGTAGTCGTAGGTGGGCATGTCG JPH3_67329 128 319 Forward GTAGGTTTAATTTTGACGACGGA primer 330Reverse GAAACCGTAACTCCACGAAC primer 331 BeaconCGACATGCGAGGACGGTAAGGCGTAC GGGCATGTCG JPH3_67330  92 319 ForwardGTAGGTTTAATTTTGACGACGGA primer 332 Reverse ACCCTTAAAACCGATACAAACG primer331 Beacon CGACATGCGAGGACGGTAAGGCGTAC GGGCATGTCG JPH3_67331  90 313Forward GGGGTAGGTTTAATTTTGACGAC primer 314 Reverse TAAAACCGATACAAACGCCAprimer 331 Beacon CGACATGCGAGGACGGTAAGGCGTAC GGGCATGTCG JPH3_67332 115333 Forward TACGGTTTAATCGGAGGACGTAG primer 334 ReverseAACGAAAATAAATACCGCGAA primer 335 Beacon CGACATGCGGGCGCGATCGGAAGTACGGCATGTCG JPH3_67333 109 333 Forward TACGGTTTAATCGGAGGACGTAG primer 336Reverse AATAAATACCGCGAACCGAA primer 335 BeaconCGACATGCGGGCGCGATCGGAAGTAC GGCATGTCG JPH3_67334  92 333 ForwardTACGGTTTAATCGGAGGACGTAG primer 337 Reverse GAACCGAACCGAAACGAAA primer335 Beacon CGACATGCGGGCGCGATCGGAAGTAC GGCATGTCG JPH3_67335  96 51Forward TTAGATTTCGTAAACGGTGAAAAC primer 52 Reverse TCTCCTCCGAAAAACGCTCprimer 338 Beacon CGACATGCGCGGTCGTCGGCGGTTTT GGCATGTCG JPH3_67336 108339 Forward TGTAATTCGGTTTTAGATTTCGT primer 52 ReverseTCTCCTCCGAAAAACGCTC primer 338 Beacon CGACATGCGCGGTCGTCGGCGGTTTTGGCATGTCG JPH3_67337  91 340 Forward GTTCGTTTTTCGTTTTTCGTTT primer 341Reverse CTAACCTACTAAACCGCGCC primer 338 BeaconCGACATGCGCGGTCGTCGGCGGTTTT GGCATGTCG JPH3_67338  97 342 ForwardGTTTTCGTTCGTTTTTCGTTT primer 341 Reverse CTAACCTACTAAACCGCGCC primer 338Beacon CGACATGCGCGGTCGTCGGCGGTTTT GGCATGTCG JPH3_67339 120 343 ForwardAGTAGTAGTAGTAATGCGGCGGT primer 344 Reverse CGAACGAACGAAATACGAAC primer345 Beacon CGACATGCGCGTTTCGGGTTCGGTTC GGCATGTCG JPH3_67340 126 346Forward GGGTAGTTTAGGTTCGGGTC primer 326 Reverse ATATAATACAACCGCCAACGCCprimer 347 Beacon CGACATGCGCGGGCGTTCGAGGGCGC ATGTCG

In specific embodiments, the methods of the invention employ or relyupon or utilise primers and/or probes selected from the primers andprobes comprising the nucleotide sequences set forth in Table 12 belowto determine the methylation status of the at least one gene. The tablepresents specific primer and probe combinations for certain preferredgenes whose methylation status may be determined according to themethods of the invention.

TABLE 12 Primer sequences and beacon (probe) sequences SEQ ID Oligo NOnucleotides BNIP3 348 forward 5′-TACGCGTAGGTTTTAAGTCGC-3′ primer 349reverse 5′-TCCCGAACTAAACGAAACCCCG-3′ primer 350 beacon 5′-FAM-CGACATGCCTACGACCGCGTCGCCCATTAGCAT GTCG-3′-DABCYL FOXE1 351 forward5′-TTTGTTCGTTTTTCGATTGTTC-3′ primer 352 reverse5′-TAACGCTATAAAACTCCTACCGC-3′ primer 353 beacon 5′-FAM-CGTCTCGTCGGGGTTCGGGCGTATTTTTTTAGG TAGGCGAGACG-3′-DABCYL JAM3 354 forward5′-GGGATTATAAGTCGCGTCGC-3′ primer 355 reverse 5′-CGAACGCAAAACCGAAATCG-3′primer 356 beacon 5′-FAM- CGACACGATATGGCGTTGAGGCGGTTATCGTGT CG-3′-DABCYLJPH3  51 forward 5′-TTAGATTTCGTAAACGGTGAAAAC-3′ primer  52 reverse5′-TCTCCTCCGAAAAACGCTC-3′ primer  53 beacon 5′-FAM-CGTCTGCAACCGCCGACGACCGCGACGCAGACG- 3′-DABCYL PHACTR3 357 forwardTTATTTTGCGAGCGGTTTC primer 358 reverse GAATACTCTAATTCCACGCGACT primer359 beacon CGACATGCGGGTTCGGTCGGCGCGGGGCATGTC G TFPI2 360 forward5′-GTTCGTTGGGTAAGGCGTTC-3′ primer 361 reverse5′-CATAAAACGAACACCCGAACCG-3′ primer 362 beacon 5′-FAM-CGACATGCACCGCGCACCTCCTCCCGCCAAGCA TGTCG-3′-DABCYL SOX17 363 forward5′-GAGATGTTTCGAGGGTTGC-3′ primer 364 reverse 5′-CCGCAATATCACTAAACCGA-3′primer 365 beacon 5′-FAM- CGACATGCGTTCGTGTTTTGGTTTGTCGCGGTTTGGCATGTCG-3′-DABCYL SYNE1 366 forward5′-GTTGGGTTTTCGTAGTTTTGTAGATCGC-3′ primer 367 reverse5′-CTACGCCCAAACTCGACG-3′ primer 368 beacon 5′-FAM-CGACATGCCCCGCCCTATCGCCGAAATCGCATG TCG-3′-DABCYL

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers and beaconscomprising the nucleotide sequences set forth in Table 13 below todetermine the methylation status of BNIP3. The table presents specificprimer combinations for determining the methylation status of this geneand the primer pairs may be selected according to the table.

TABLE 13Additional assay designs: Primer and probe sequences for determining themethylation status of BNIP3, with predicted amplification productlengths shown. Ampli- SEQ 5′ to 3′ Sequences (all con ID Oligothe beacons are 5′-FAM and Assay name length NO nucleotides 3′-DABCYL)BNIP3_13409  94 369 Forward AGTGTTTAGAGAGTTCGTCGGTT primer 370 ReverseCGTAACGAATAAACTACGCGAT primer 371 Beacon CGACATGCGGAGAATTCGGTTTATCGTTCGTCGCGCATGTCG BNIP3_67227 159 372 Forward TTTTAGGTGGAATTTTAGTTCGC primer373 Reverse CCCTCCTACGAACATACGAAA primer 374 BeaconCGACATGCCGTGCGGTTCGATTCGGGTTT AAGGCATGTCG BNIP3_67229 160 375 ForwardCGGTTTAATTGCGAGACGTAG primer 376 Reverse AACGTAAAAACCCCGCGTA primer 377Beacon CGACATGCCGTGCGGTTCGATTCGGGCAT GTCG BNIP3_67231 107 378 ForwardGTTTTCGGGTTTTTGTTCGT primer 379 Reverse GACTCTACTCGAACCTCCGCT primer 380Beacon CGACATGCGGGCGTTCGTTCGTAGGAAGA AGGCATGTCG BNIP3_67232 141 381Forward TGAGGACGTGTAGGGAAGC primer 382 Reverse AAACGAACAAAAACCCGAAAprimer 383 Beacon CGACATGCCGAGCGGTGGGTCGGAGGCAT GTCG BNIP3_67233 153 384Forward GCGTTAGAGGGTAATTGCG primer 385 Reverse CTATAAATTCCTCCGACCGAACprimer 386 Beacon CGACATGCCGCGTCGGGTTGCGGGCATGT CG BNIP3_67235  94 387Forward TTTGTATTTCGGGCGTTTC primer 388 Reverse GCAACTAAAACACATCCCGCprimer 389 Beacon CGACATGCGCGATATGGCGTTAGAGGGTA ATTGCGCATGTCGBNIP3_67236 106 390 Forward GGTTTTTACGGAAGTCGGG primer 391 ReverseAATACAAACGCGATATAAAACGAA primer 392 Beacon CGACATGCGCGTTATTTCGTTTCGTGGACGGGCATGTCG BNIP3_67239 151 393 Forward GATTTCGCGTATTGTTCGG primer 394Reverse GATCCAACTACGAAACGCA primer 395 BeaconCGACATGCGGTTTGGATTCGGGTCGGATC GGCATGTCG

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers and beaconscomprising the nucleotide sequences set forth in Table 14 below todetermine the methylation status of FOXE1. The table presents specificprimer combinations for determining the methylation status of this geneand the primer pairs may be selected according to the table.

TABLE 14Additional assay designs: Primer and probe sequences for determining themethylation status of FOXE1, with predicted amplification product lengths shown.Ampli- SEQ 5′ to 3′ Sequences (all con ID Oligothe beacons are 5′-FAM and Assay name length NO nucleotides 3′-DABCYL)FOXE1_13297 108 396 Forward TTCGTTTCGAGAAGTATTACGC primer 397 ReverseGCGCTAAAAACTCAACGTCC primer 398 Beacon CGACATGCGAGTCGTCGGTTAGCGGGTTATTTTCGGCATGTCG FOXE1_13307 133 399 Forward TTCGTTTTCGGTAGTTATGGCprimer 400 Reverse GATCCCCTAAACTCTCCGC primer 401 BeaconCGACATGCCGGGTTTTGGATTTTCGC GGTTGTCGGCATGTCG FOXE1_13317 111 402 ForwardCGGAGAGTTTAGGGGATCGT primer 403 Reverse CTCTATCTACACCGCGCCA primer 404Beacon CGACATGCGTTTAGGTTGGTACGCGT TGGAGGGCATGTCG FOXE1_67265 118 405Forward ATCGGTGTCGTTTTACGTTTC primer 406 Reverse GTAAATCTCCAACCCTACGAACprimer 407 Beacon CGACATGCGCGGAGGGAGGAGTCGGG CATGTCG FOXE1_67266 125 408Forward TAGGGAATCGGTGTCGTTTTAC primer 409 ReverseCGTAAATCTCCAACCCTACGAAC primer 410 Beacon CGACATGCCGGAGGGAGGAGTCGGTTCGGGCATGTCG FOXE1_67267 108 411 Forward TGAGGTTTTTCGAGTCGGTT primer 412Reverse CCACAACGTCAAAACGAAA primer 413 Beacon CGACATGCCGGGTTTTAGTCGATCGGGGCATGTCG FOXE1_67268 100 414 Forward ACGTTCGCGTTATGATTGTC primer 415Reverse CCGACCCCTACTACCGTCT primer 416 Beacon CGACATGCCGTAGTCGGAGGTGTTGGTTATCGGCATGTCG FOXE1_67270 124 417 Forward GAGGTTATCGTCGTTGTTCGT primer397 Reverse GCGCTAAAAACTCAACGTCC primer 418 BeaconCGACATGCCGCGGGTTGAGTCGTCGG GCATGTCG FOXE1_67271 116 419 ForwardTTAGGGATTATTTTCGGATTTTTC primer 420 Reverse TTCTCGAAACGAACAACGAC primer421 Beacon CGACATGCCGTTCGGTATTAGCGCGT AAGGGGCATGTCG FOXE1_67274  92 422Forward CGGTAGAAGGGGAAGCGTT primer 423 Reverse CTCATCGCCATAACCATCGprimer 424 Beacon CGACATGCGCGTGAGGCGGCGTTCGG CATGTCG FOXE1_67276  90 351Forward TTTGTTCGTTTTTCGATTGTTC primer 425 Reverse CTATAAAACTCCTACCGCGCCprimer 426 Beacon CGACATGCCGGGGTTCGGGCGTATTT TTTTAGGGCATGTCG FOXE1_67278 98 427 Forward TGTGCGCGTAGAAGAGGTTTC primer 428 ReverseCGAAAACAAAACATAAACGACC primer 429 Beacon CGACATGCGGTTAGAGCGAGGGTAGTTAGTATTGGGCATGTCG FOXE1_67279  90 430 Forward GTGCGCGTAGAAGAGGTTTCprimer 431 Reverse AAAACATAAACGACCCCCG primer 432 BeaconCGACATGCGAGCGAGGGTAGTTAGTA TTGGCGGCATGTCG

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers and beaconscomprising the nucleotide sequences set forth in Table 15 below todetermine the methylation status of JAM3. The table presents specificprimer combinations for determining the methylation status of this geneand the primer pairs may be selected according to the table.

TABLE 15Additional assay designs: Primer and probe sequences for determining themethylation status of JAM3, with predicted amplification product lengthsshown. Ampli- SEQ Oligo 5′ to 3′ Sequences (all Assay con ID nucleotidesthe beacons are 5′-FAM and name length NO & probes 3′-DABCYL) JAM3_ 104433 Forward TGTGTCGGTTTAGAGTATCGTTG 12721 primer 434 ReverseCAATTACCATAACGACCGCC primer 435 BeaconCGACATGCGTTATTATGGTGTCGGTTCGGTTGGG CATGTCG JAM3_ 108 433 ForwardTGTGTCGGTTTAGAGTATCGTTG 67314 primer 436 Reverse GCCCCAATTACCATAACGACCprimer 435 Beacon CGACATGCGTTATTATGGTGTCGGTTCGGTTGGG CATGTCG JAM3_ 113437 Forward ATTTATGTGTCGGTTTAGAGTATCG 67315 primer 436 ReverseGCCCCAATTACCATAACGACC primer 435 BeaconCGACATGCGTTATTATGGTGTCGGTTCGGTTGGG CATGTCG JAM3_  90 438 ForwardTCGAGTTTTAGTTTTGGTTGC 67317 primer 439 Reverse AAATAACGATCCTAACTCCGAAAprimer 440 Beacon CGACATGCCGGTTCGGGATTTCGGGAGGCATGTC G JAM3_ 133 441Forward TTTAGTAAGTTTTAGCGTTTACGTC 67318 primer 442 ReverseGAATAAACTCCTCCCAAACGAA primer 443 BeaconCGACATGCGAGGGTCGTGTTTATCGTTCGGGCAT GTCG

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers and beaconscomprising the nucleotide sequences set forth in Table 16 below todetermine the methylation status of PHACTR3. The table presents specificprimer combinations for determining the methylation status of this geneand the primer pairs may be selected according to the table.

TABLE 16Additional assay designs: Primer and probe sequences for determining themethylation status of PHACTR3, with predicted amplification productlengths shown. Ampli- SEQ Oligo 5′ to 3′ Sequences (all Assay con IDnucleotides the beacons are 5′-FAM and name length NO & probes3′-DABCYL) PHACTR3_ 111 444 Forward ATTTAGGTAACGGGTTGGGC 67295 primer445 Reverse ACTCCCCGAATACAAACGAA primer 446 BeaconCGACATGCGGTTCGAGGTAGGTGGCGTT GGCATGTCG PHACTR3_ 128 447 ForwardTTCGTAGAGTGATTTTAGCGTTT 67296 primer 448 Reverse AACGCCACCTACCTCGAACprimer 449 Beacon CGACATGCGCGGACGTCGGGGAGAATTT AGGGCATGTCG PHACTR3_  92450 Forward TAATTTGTTTTCGCGTCGG 67297 primer 451 ReverseCTAAAATCACTCTACGAACGACC primer 452 Beacon CGACATGCGGACGGGAGCGGTTGTTTCGGCATGTCG PHACTR3_ 118 453 Forward CGTTTCGGATGTTTTGATTTTAC 67298 primer454 Reverse ACTCTACGAACGACCCCGC primer 455 BeaconCGACATGCCGGAGGACGGGAGCGGGCAT GTCG PHACTR3_ 136 456 ForwardTTCGTCGGTGATTTTGGTC 67299 primer 454 Reverse ACTCTACGAACGACCCCGC primer457 Beacon CGACATGCCGTCGGTCGGGTTTATGGTC GCATGTCG PHACTR3_ 128 458Forward ACGTTGTTACGAAATCGGG 67302 primer 459 ReverseAAACGCCTAACTCCAACGAAA primer 460 Beacon CGACATGCGGCGTACGTTTTTCGTTTTTTTGTCGGCGGCATGTCG PHACTR3_ 118 458 Forward ACGTTGTTACGAAATCGGG 67303primer 461 Reverse CTCCAACGAAACCTAACGCA primer 460 BeaconCGACATGCGGCGTACGTTTTTCGTTTTT TTGTCGGCGGCATGTCG PHACTR3_ 110 462 ForwardCGTTGTTACGAAATCGGGT 67304 primer 463 Reverse GAAACCTAACGCACCTAAACGprimer 460 Beacon CGACATGCGGCGTACGTTTTTCGTTTTT TTGTCGGCGGCATGTCGPHACTR3_ 103 462 Forward CGTTGTTACGAAATCGGGT 67305 primer 464 ReverseAACGCACCTAAACGCGCTA primer 460 Beacon CGACATGCGGCGTACGTTTTTCGTTTTTTTGTCGGCGGCATGTCG PHACTR3_  93 465 Forward GATACGAGGTAGTCGTTTTCGTT 67306primer 358 Reverse GAATACTCTAATTCCACGCGACT primer 466 BeaconCGACATGCGCGGTTATGGGTTCGGTCGG GCATGTCG PHACTR3_ 124 467 ForwardGACGTTGGGGTTATTTTGC 67308 primer 358 Reverse GAATACTCTAATTCCACGCGACTprimer 468 Beacon CGACATGCGCGATACGAGGTAGTCGTTT TCGTTTTTCGGCATGTCGPHACTR3_  92 469 Forward CGTCGTTTTCGTTTAGTTCGT 67309 primer 470 ReverseGCAAAATAACCCCAACGTCC primer 471 Beacon CGACATGCGCGGAGGAGGTGGTCGAGGCATGTCG PHACTR3_ 133 472 Forward GATTGGGGATAGGAATCGC 67310 primer 473Reverse AACGACGAACGAATCGAAA primer 471 BeaconCGACATGCGCGGAGGAGGTGGTCGAGGC ATGTCG PHACTR3_ 113 472 ForwardGATTGGGGATAGGAATCGC 67311 primer 474 Reverse AACCCGAAACAAATAACGCT primer475 Beacon CGACATGCGCGGTTTTTCGAATGTAGGC GGGCATGTCG PHACTR3_ 101 472Forward GATTGGGGATAGGAATCGC 67312 primer 476 ReverseATAACGCTAAAAACAAAACCCCG primer 475 Beacon CGACATGCGCGGTTTTTCGAATGTAGGCGGGCATGTCG PHACTR3_  92 472 Forward GATTGGGGATAGGAATCGC 67313 primer 477Reverse AAAACAAAACCCCGCGAAA primer 475 BeaconCGACATGCGCGGTTTTTCGAATGTAGGC GGGCATGTCG

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers and beaconscomprising the nucleotide sequences set forth in Table 17 below todetermine the methylation status of TFPI2. The table presents specificprimer combinations for determining the methylation status of this geneand the primer pairs may be selected according to the table.

TABLE 17 Additional assay designs: Primer and probe sequences fordetermining the methylation status of TFPI2, with predictedamplification product lengths shown. Ampli- SEQ 5′ to 3′ Sequences (allAssay con ID Oligo the beacons are 5′-FAM and name length NO nucleotides3′-DABCYL) TFPI2_ 117 478 Forward CGGGGTGATAGTTTTCGTG 12620 primer 479Reverse CGACTTTCTACTCCAAACGACC primer 480 BeaconCGACATGCGGGTCGGTCGGACGTTCG GCATGTCG TFPI2_  98 481 ForwardTAGAAATTGTTGGCGTTGTTTTC 67243 primer 482 Reverse TACCGAACCCTACTTCTCCGTprimer 483 Beacon CGACATGCCGTATAGGAATTGGCGGT AGTTTTGCGTGGCATGTCG TFPI2_124 484 Forward TAGTCGTCGGCGTAAGGAGC 67244 primer 485 ReverseAAAACTACGAAAACAACGCCA primer 486 Beacon CGACATGCTGGGTGCGCGTAGGGTAGCATGTCG TFPI2_ 120 487 Forward GTGTTCGTTTTATGCGGGG 67245 primer 488Reverse TCTTACACAATTTACAACGCGAA primer 489 BeaconCGACATGCCGTTCGGTCGATTTTCGT CGGGCATGTCG TFPI2_ 115 490 ForwardTTTTTGTTTTAGGCGGTTC 67246 primer 491 Reverse GACGAAATAACAATCCCCGT primer489 Beacon CGACATGCCGTTCGGTCGATTTTCGT CGGGCATGTCG TFPI2_ 106 492 ForwardTTCGTTAGGAAAAGTAGTAGAATCG 67247 primer 493 Reverse GCCAAACGCTTTCTCGAACprimer 494 Beacon CGACATGCGGGTAAGGCGTTCGAGAA AGCGGCATGTCG TFPI2_ 117 478Forward CGGGGTGATAGTTTTCGTG 67248 primer 479 ReverseCGACTTTCTACTCCAAACGACC primer 495 Beacon CGACATGCGTCGGTCGGACGTTCGTTTCGGCATGTCG TFPI2_ 120 496 Forward GTCGTTAGTTTTTGTACGGGG 67250 primer497 Reverse GAAAATCCTAAATACGCGCAA primer 498 BeaconCGACATGCGGGAGGTTTGCGACGATG TTTGTTGGGCATGTCG

In a further specific embodiment, the methods of the invention employ orrely upon or utilise primers selected from the primers and beaconscomprising the nucleotide sequences set forth in Table 18 below todetermine the methylation status of SOX17. The table presents specificprimer combinations for determining the methylation status of this geneand the primer pairs may be selected according to the table.

TABLE 18Additional assay designs: Primer and probe sequences for determining themethylation status of SOX17, with predicted amplification product lengths shown. Ampli- SEQ 5′ to 3′ Sequences (all Assay con ID Oligothe beacons are 5′-FAM and name length NO nucleotides 3′-DABCYL) SOX17_117 499 Forward GGCGTTAGAGTTTAGTTTCGGT 66067 primer 500 ReverseTAATCCGAATCCCACGTCC primer 501 Beacon CGACATGCGGTGTAGTTTTGGGCGCGGGCATGTCG SOX17_ 131 502 Forward CGGTTTAGTGATATTGCGGG 66070 primer 503Reverse ACGTAAAACTCGAACCACGAC primer 504 Beacon CGACATGCGATGTGGTTAATGGAGCGGCGAGGGCATGTCG SOX17_ 110 505 Forward TTAGTGATATTGCGGGCGT 66071 primer506 Reverse CGACCTAAACGTAAACCTAACGA primer 507 BeaconCGACATGCGGAGCGGCGAGGGCGG CATGTCG SOX17_  92 508 ForwardTATTGAGATGTTTCGAGGGTTGC 66073 primer 509 ReverseCTAAATACGCTATAAACCAAACCG primer 510 Beacon CGACATGCCGGTTCGAAGTCGTCGTTCGTGGCATGTCG SOX17_  96 511 Forward TCGAGTTAAGGGCGAGTTTC 66078 primer512 Reverse TCTAAATTCTACTACGCCAACCG primer 513 BeaconCGACATGCGGTGTGGGTTAAGGAC GAGCGTAAGGCATGTCG SOX17_  91 511 ForwardTCGAGTTAAGGGCGAGTTTC 66079 primer 514 Reverse ATTCTACTACGCCAACCGCTprimer 515 Beacon CGACATGCCGGCGGTCGATGAACG TTTTTATGGGCATGTCG SOX17_ 117516 Forward CGAATAGCGGAGTATCGGTC 66080 primer 517 ReverseACTACGCCAACCGCTTACG primer 518 Beacon CGACATCGCGGGTCGAGTTAAGGG CGATGTCGSOX17_ 119 519 Forward TTTAGTATTTTGTTTAATTCGGCG 66082 primer T 520Reverse AACGAATCCCGTATCCGAC primer 521 Beacon CGACATGCGGATTTTGTTGCGTTAGTCGTTTGCGTTCGCATGTCG

Each and all of these primers and probes form separate aspects of theinvention. In particular, the invention relates to primer pairs selectedfrom the primer pairs disclosed herein, including in the tables (whichmay comprise additional sequence over above the basic sequence listed).Further characteristics of these primers are summarized in the detaileddescription (experimental part) below. It is noted that variants ofthese sequences may be utilised in the present invention. In particular,additional sequence specific flanking sequences may be added, forexample to improve binding specificity, as required. Variant sequencespreferably have at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% nucleotide sequence identity with the nucleotide sequencesof the primers and/or probes set forth in any of tables 2, 3, 4, 5 or 6.The primers and probe (including hairpin) structures may incorporatesynthetic nucleotide analogues as appropriate or may be RNA or PNA basedfor example, or mixtures thereof.

Similarly alternative fluorescent donor and acceptor moieties/FRET pairsmay be utilised as appropriate. In addition to being labelled with thefluorescent donor and acceptor moieties, the primers may includemodified oligonucleotides and other appending groups and labels providedthat the functionality as a primer in the methods of the invention isnot compromised. Similarly alternative fluorescent donor and acceptormoieties/FRET pairs may be utilised as appropriate. Molecules that arecommonly used in FRET include fluorescein, 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), and 5-(2′-aminoethyl) aminonaphthalene-1-sulfonicacid (EDANS). Whether a fluorophore is a donor or an acceptor is definedby its excitation and emission spectra, and the fluorophore with whichit is paired. For example, FAM is most efficiently excited by light witha wavelength of 488 nm, and emits light with a spectrum of 500 to 650nm, and an emission maximum of 525 nm. FAM is a suitable donorfluorophore for use with JOE, TAMRA, and ROX (all of which have theirexcitation maximum at 514 nm).

Thus, in one embodiment, said donor moiety and said acceptor moiety areselected from 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′-tetramethyl-6-carboxyrhodamine (TAMRA),6-carboxy-X-rhodamine (ROX), 5-(2′-aminoethyl)aminonapthalene-1-sulfonicacid (EDANS), anthranilamide, coumarin, terbium chelate derivatives,Malachite green, Reactive Red 4, DABCYL, tetramethyl rhodamine, pyrenebutyrate, eosine nitrotyrosine, ethidium, and Texas Red. In a furtherembodiment, said donor moiety is selected from fluorescein,5-carboxyfluorescein (FAM), rhodamine,5-(2′-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS),anthranilamide, coumarin, terbium chelate derivatives, Malachite green,and Reactive Red 4, and said acceptor moiety is selected from DABCYL,rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine,ethidium, and Texas Red.

In one particular embodiment, said donor moiety is fluorescein or aderivative thereof, and said acceptor

moiety is DABCYL. In specific embodiments, the fluorescein derivativecomprises, consists essentially of or consists of 6-carboxy fluorescein.

For all aspects and embodiments of the invention, the primers and inparticular the stem loop/hairpin structures, and/or the probes (asappropriate upon the form of detection employed) may be labelled withdonor and acceptor moieties during chemical synthesis of the primers orprobes or the label may be attached following synthesis using anysuitable method. Many such methods are available and well characterisedin the art.

It is noted that the specific exemplified probe types (such as thehairpin probe type employed in tables 7 and 9) may be replaced asappropriate with a different probe (or primer) type as appropriate.Equivalents are discussed herein and may be utilised as appropriate.

In a further embodiment, bisulphite sequencing is utilised in order todetermine the methylation status of the at least one gene selected froman NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein). Primers may be designed for use insequencing through the important CpG islands in the at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein). Thus, primers may bedesigned in both the sense and antisense orientation to directsequencing across the promoter region of the relevant gene or genes.

In one embodiment, in which the NDRG4 and/or NDRG2 gene is sequenced,bisulphite sequencing may be carried out by using sequencing primerswhich comprise, consist essentially of or consist of the followingsequences, and which may be used in isolation or in combination tosequence both strands:

NDRG4 primers SEQ ID NO: 570 5′-gatyggggtgttttttaggttt-3′ (forward)wherein ″Y″ represents a pyrimidine mucleotide SEQ ID NO: 65′-craacaaccaaaaacccctc-3′ (reverse) Wherein ″r″represents a purine nucleotide. NDRG2 primers SEQ ID NO: 5225′-tttgttggttattttttttttattttt-3′ (forward) SEQ ID NO: 5235′-cccccaaactcaataataaaaac-3′ (reverse)

These sequencing primers form a further aspect of the invention, withsuitable variants being included within the scope of the invention (thediscussion of which applies mutatis mutandis here).

Other nucleic acid amplification techniques, in addition to PCR (whichincludes real-time versions thereof and variants such as nested PCR),may also be utilised, as appropriate, to detect the methylation statusof the at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein).Such amplification techniques are well known in the art, and includemethods such as NASBA (Compton, 1991), 3SR (Fahy et al., 1991) andTranscription Mediated Amplification (TMA). Other suitable amplificationmethods include the ligase chain reaction (LCR) (Barringer et al, 1990),selective amplification of target polynucleotide sequences (U.S. Pat.No. 6,410,276), consensus sequence primed polymerase chain reaction(U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction(WO 90/06995), invader technology, strand displacement technlology, andnick displacement amplification (WO 2004/067726). This list is notintended to be exhaustive; any nucleic acid amplification technique maybe used provided the appropriate nucleic acid product is specificallyamplified. Thus, these amplification techniques may be tied in to MSPand/or bisulphite sequencing techniques for example.

Sequence variation that reflects the methylation status at CpGdinucleotides in the original genomic DNA offers two approaches toprimer design. Both primer types may be utilised in the methods of theinvention either alone or in combination. Firstly, primers may bedesigned that themselves do not cover any potential sites of DNAmethylation. Sequence variations at sites of differential methylationare located between the two primers. Such primers are used in bisulphitegenomic sequencing, COBRA and Ms-SnuPE for example. Secondly, primersmay be designed that anneal specifically with either the methylated orunmethylated version of the converted sequence. If there is a sufficientregion of complementarity, e.g., 12, 15, 18, or 20 nucleotides, to thetarget, then the primer may also contain additional nucleotide residuesthat do not interfere with hybridization but may be useful for othermanipulations. Examples of such other residues may be sites forrestriction endonuclease cleavage, for ligand binding or for factorbinding or linkers or repeats. The oligonucleotide primers may or maynot be such that they are specific for modified methylated residues.

One way to distinguish between modified and unmodified DNA is tohybridize oligonucleotide primers which specifically bind to one form orthe other of the DNA. After hybridization, an amplification reaction canbe performed and amplification products assayed. The presence of anamplification product indicates that a sample hybridized to the primer.The specificity of the primer indicates whether the DNA had beenmodified or not, which in turn indicates whether the DNA had beenmethylated or not.

Another way to distinguish between modified and unmodified DNA is to useoligonucleotide probes which may also be specific for certain products.Such probes may be hybridized directly to modified DNA or toamplification products of modified DNA. Oligonucleotide probes can belabelled using any detection system known in the art. These include butare not limited to fluorescent moieties, radioisotope labelled moieties,bioluminescent moieties, luminescent moieties, chemiluminescentmoieties, enzymes, substrates, receptors, or ligands.

In the MSP technique, amplification is achieved with the use of primersspecific for the sequence of the gene whose methylation status is to beassessed. In order to provide specificity for the nucleic acidmolecules, primer binding sites corresponding to a suitable region ofthe sequence may be selected. The skilled reader will appreciate thatthe nucleic acid molecules may also include sequences other than primerbinding sites which are required for detection of the methylation statusof the gene, for example RNA Polymerase binding sites or promotersequences may be required for isothermal amplification technologies,such as NASBA, 3SR and TMA.

TMA (Gen-probe Inc.) is an RNA transcription amplification system usingtwo enzymes to drive the reaction, namely RNA polymerase and reversetranscriptase. The TMA reaction is isothermal and can amplify either DNAor RNA to produce RNA amplified end products. TMA may be combined withGen-probe's Hybridization Protection Assay (HPA) detection technique toallow detection of products in a single tube. Such single tube detectionis a preferred method for carrying out the invention.

Whilst the genes (in particular promoters) of the invention appear to beunmethylated in normal tissues, and thus the detection of methylation(or indeed a lack of methylation) in these genes is readily observableas being significant in terms of a cancer diagnosis and also inselecting suitable treatment regimens and for determining the likelihoodof successful treatment or resistance to treatment with certainanti-cancer agents etc, when determining methylation status, it may bebeneficial to include suitable controls in order to ensure the methodchosen to assess this parameter is working correctly and reliably. Forexample, suitable controls may include assessing the methylation statusof a gene known to be methylated. This experiment acts as a positivecontrol to ensure that false negative results are not obtained (i.e. aconclusion of a lack of methylation is made even though the at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) may, in fact, bemethylated). The gene may be one which is known to be methylated in thesample under investigation or it may have been artificially methylated,for example by using a suitable methyltransferase enzyme, such as SssImethyltransferase. In one specific embodiment, the NDRG4/NDRG2 subfamilygene, preferably the NDRG4 and/or NDRG2 gene, may be assessed in normallymphocytes, following treatment with SssI methyltransferase, as apositive control.

Additionally or alternatively, suitable negative controls may beemployed with the methods of the invention. Here, suitable controls mayinclude assessing the methylation status of a gene known to beunmethylated or carrying out an amplification in the absence of DNA (forexample by using a water only sample). The former experiment acts as anegative control to ensure that false positive results are not obtained(i.e. a conclusion of methylation is made even though the at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein, such as at least onegene selected from OSMR, SFRP1, NDRG4, GATA5, ADAM23, JPH3, SFRP2 andAPC in one specific embodiment) may, in fact, be unmethylated). The genemay be one which is known to be unmethylated in the sample underinvestigation or it may have been artificially demethylated, for exampleby using a suitable DNA methyltransferase inhibitor, such as thosediscussed in more detail below. In one specific embodiment, theNDRG4/NDRG2 subfamily gene, in particular the NDRG4 and/or NDRG2 gene,may be assessed in normal lymphocytes as a negative control, since ithas been shown for the first time herein that the NDRG4 and/or NDRG2gene is unmethylated in normal tissues.

The application of the methods of present invention to extremely smallamounts of abnormally-methylated DNA, that are released into collectedfluids, in particular stools, may require the generation andamplification of a DNA library before testing for methylation of anyspecific gene. Suitable methods on whole genome amplification andlibraries generation for such amplification (e.g. Methylplex andEnzyplex technology, Rubicon Genomics) are described in US2003/0143599,WO2004/081225 and WO2004/081183 for example. In addition, WO2005/090507describes library generation/amplification methods that require eitherbisulfite conversion or non-bisulfite based application. Bisulfitetreatment may occur before or after library construction and may requirethe use of adaptors resistant to bisulfite conversion. Meth-DOP-PCR (DiVinci et al, 2006), a modified degenerate oligonucleotide-primed PCRamplification (DOP-PCR) that is combined with MSP, provides anothersuitable method for specific detection of methylation in small amountsof DNA. Improved management of patient care may require these existingmethods and techniques to supplement the methods of the invention.

As discussed in the experimental section, epigenetic silencing resultingin methylation of the NDRG4/NDRG2 subfamily gene has been shown in anumber of gastrointestinal cancers such as colorectal cancer and/orgastric cancer, stomach and oesophageal cancers, in particularoesophageal carcinomas. Thus, in specific embodiments, the inventionprovides for a method of diagnosing a gastrointestinal cancer, such ascolorectal cancer and/or gastric cancer and/or oesophageal cancer orpredisposition to a gastrointestinal cancer, such as colorectal cancerand/or gastric cancer and/or oesophageal cancer comprising detecting themethylation status of the NDRG4/NDRG2 subfamily gene, whereinmethylation of the gene is indicative for a gastrointestinal cancer,such as colorectal cancer and/or gastric cancer and/or oesophagealcancer, or predisposition to a gastrointestinal cancer, such ascolorectal cancer and/or gastric cancer and/or oesophageal cancer.Preferably, the gene is NDRG2, or NDRG4, or a combination of NDRG2 andNDRG4.

Whilst the epigenetic change, in particular methylation status, of anyof at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein, suchas at least one gene selected from GATA4, OSMR, NDRG4, GATA5, SFRP1,ADAM23, JPH3, SFRP2, APC and MGMT) may be determined in order todiagnose a gastrointestinal cancer, such as colorectal cancer and/orgastric cancer and/or oesophageal cancer or a predisposition thereto. Inspecific embodiments, the at least one gene may be selected from GATA4,OSMR, NDRG4 and SFRP2, in particular where faecal samples are utilized.Detecting an epigenetic change, in particular methylation, in thesegenes results in a particularly sensitive and specific diagnosticmethod. In a further embodiment, where plasma or serum samples areutilised, the at least one gene may be selected from OSMR, SFRP1, NDRG4,GATA5, ADAM23, JPH3, SFRP2 and APC and particularly selected from OSMR,NDRG4, GATA5 and ADAM23, in particular where plasma or serum samples areutilised. Detecting an epigenetic change, in particular methylation, inthese genes results in a particularly sensitive and specific diagnosticmethod.

Additionally or alternatively, the at least one gene may be selectedfrom TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3, such as fromTFPI2, BNIP3, FOXE1, SYNE1 and SOX17, in particular TFPI2.

In embodiments in which tissue samples are utilised, the methods maycomprise, consist essentially of or consist of detecting an epigeneticchange in a panel of genes comprising OSMR, GATA4 and ADAM23 or OSMR,GATA4 and GATA5, wherein detection of the epigenetic change in at leastone of the genes in the panel is indicative of a predisposition to, orthe incidence of, colorectal cancer. The tissue sample may comprise,consist essentially of or consist of a colon and/or rectal and/orappendix sample for example, as discussed herein above.

In embodiments where faecal samples are employed, the at least one genemay be selected from GATA4, OSMR, NDRG4, GATA5, SERP1, ADAM23, JPH3,SFRP2, APC and MGMT. in addition, or alternatively, the at least onegene may be selected from TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3,and JAM3, such as from TFPI2, FOXE1, SYNE1, SOZ17, PHACTR3 and JAM3, inparticular TFPI2.

Moreover, in order to improve the sensitivity of the methods of theinvention the methods may comprise detecting an epigenetic change in apanel of genes comprising at least two, three, four, five or six of thegenes, wherein detection of an epigenetic change in at least one of thegenes in the panel is indicative of a predisposition to, or theincidence of, cancer and in particular gastrointestinal cancers asdefined herein, such as colorectal cancer. The panel of genes maycomprise/consist essentially of or consist of two, three, four, five orsix genes.

Certain panels of genes have been found to result in particularlysensitive methods for detecting a gastrointestinal cancer, such ascolorectal cancer and/or gastric cancer and/or oesophageal cancer or apredisposition thereto—especially colorectal cancer. Accordingly, in oneembodiment, the panel of genes comprises, consists essentially of orconsists of GATA4 and OSMR, GATA4 and NDRG4, GATA4 and SFRP2, OSMR andNDRG4, OSMR and SFRP2, NDRG4 and SFRP2, APC and SFRP2, APC and OSMR, APCand GATA4, APC and NDRG4, MGMT and OSMR, MGMT and GATA4, MGMT and NDRG4,MGMT and SFRP2, MGMT and APC, SFRP1 and MGMT, SFRP1 and OSMR, SFRP1 andGATA4, SFRP1 and NDRG4, SFRP1 and SFRP2, SFRP1 and APC, GATA5 and SFRP1,GATA5 and MGMT, GATA5 and OSMR, GATA5 and GATA4, GATA5 and NDRG4, GATA5and SFRP2 or GATA5 and APC. Suitable panels incorporating other genessuch as ADAM23 and/or JPH3 are also envisaged in the present invention.These embodiments are of particular applications to faecal test samples.

Further useful panels of genes comprise, consist essentially of orconsists of SFRP1, SFRP2 and APC or SFRP2, OSMR and APC. Further panelsof genes comprise, consist essentially of or consist of GATA4, OSMR andNDRG4, GATA4, OSMR and SFRP2, GATA4, NDRG4 and SFRP2 or OSMR, NDRG4 andSFRP2. One specific four gene panel consists of GATA4, OSMR, NDRG4 andSFRP2. One specific panel of at least six genes comprises, consistsessentially of or consists of NDRG4, OSMR, SFRP1, ADAM23, GATA5 andMGMT. These panels may usefully be applied to faecal test samples incertain embodiments.

In a further specific embodiment, the panel of genes comprises, consistsessentially of or consists of OSMR, GATA4 and ADAM23 or OSMR, GATA4 andGATA5. This embodiment applies in particular to tissue samples, whichmay be colon, rectal or appendix samples for example, as discussedherein.

In certain embodiments, the panel of genes comprises, consistsessentially of or consists of OSMR, NDRG4, GATA5 and ADAM23, where bloodbased samples and in particular plasma or serum samples are utilised.

Thus, the invention provides a method of detecting a predisposition to,or the incidence of, early stage colorectal cancer and in particularstage 0 to II colorectal cancer in a blood sample, or derivative thereofsuch as a plasma or serum sample (preferably a plasma sample) comprisingdetecting an epigenetic change in at least one gene selected from OSMR,NDRG4, GATA5 and ADAM23, wherein detection of the epigenetic change isindicative of a predisposition to, or the incidence of, early stagecolorectal cancer and in particular stage 0 to II colorectal cancer.This method may be applied to a panel consisting of these four genes.

It is noted that for each gene, it may be possible to detect anepigenetic change, in particular methylation of the gene, in a pluralityof locations within the same gene. Thus, for example, a gene mayincorporate more than one CpG island, or multiple sites within the sameCpG island may be investigated as appropriate. As shown in the detaileddescription (experimental part) below, for example, OSMR can be assessedat two discrete locations, both providing useful diagnostically relevantresults. The respective targets are designated herein as OSMR3 andOSMR4. In one embodiment, the panel of genes comprises, consistsessentially of or consists of both OSMR3 and OSMR4. When OSMR isreferred to herein, as for all other genes, reference is made to aninvestigation of an epigenetic change, in particular methylation whichis relevant to colorectal cancer. Thus, the panels of genes in thepresent invention may incorporate assessment of multiple sites withinthe same gene as appropriate. Primers investigating multiple siteswithin the same genes are set forth in the tables above, seeparticularly tables 2 to 18 (and especially tables 5 to 11 and 13 to18).

As discussed in greater detail herein, the detection of an epigeneticchange in each of the panel of genes may be carried out in a singlereaction. Many suitable techniques allowing multiplexing are availableand may be utilised in the present invention. Most depend upon use ofsuitable fluorescent molecules having distinguishable emission spectra.The skilled person can readily select from the many fluorophoresavailable to determine which can be used in a multiplexing context.

In one embodiment, a universal quencher is utilised together withsuitable fluorophore donors each having a distinguishable emissionwavelength maximum. A particularly useful quencher is DABCYL. Togetherwith a suitable quencher such as DABCYL the following fluorophores mayeach be utilised to allow multiplexing: Coumarin (emission maximum of475 nm), EDANS (491 nm), fluorescein (515 nm), Lucifer yellow (523 nm),BODIPY (525 nm), Eosine (543 nm), tetramethylrhodamine (575 nm) andtexas red (615 nm) (Tyagi et al., Nature Biotechnology, Vol. 16, Jan.1998; 49-53).

It is noted that the methylation status of additional genes may also bedetermined in order to supplement the methods of the invention. No genehas been found to be epigenetically silenced in every similar tumour.For this reason, it may be advantageous to target multiple DNAalterations to attain high rates of tumour detection. Thus, in oneembodiment of the methods of the invention, the methylation status ofthe at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) isanalysed in combination with the methylation status of at least oneother gene involved in the establishment of cancer. The at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) may be combined withat least two other genes involved in the establishment of cancer. The atleast one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) maybe combined with and at least three, four, five or six other genesinvolved in the establishment of cancer. For colorectal cancer, theother genes involved in the establishment of cancer may be selected fromthe group consisting of SFRP1, SFRP2, GATA-4, GATA-5, CHFR, APC(2),MGMT, p16, Vimentin, p14, RASSF1a, RAB32, SEPTIN-9, RASSF2A, TMEFF2,NGFR or SMARCA3. However, any gene involved in the establishment ofcolorectal cancer may be utilized in combination with the at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) in the methods ofpresent invention.

Genes that become methylated early in the process of carcinogenesis arenot only ideal for screening purposes, but also interesting targets forearly cancer detection and for monitoring the progression or outcome ofcancers. In a further aspect, the invention provides for a method ofcancer prognosis (prognosis to cancer) comprising detecting epigeneticsilencing of at least one gene selected from an NDRG2/NDRG4 subfamilygene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3,SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3(in all permutations and combinations including panels as discussedherein), wherein epigenetic silencing of the gene is indicative forcancer development. Preferably, epigenetic silencing is detected bydetermination of the methylation status and/or measurement of expressionlevel of the at least one gene selected from an NDRG2/NDRG4 subfamilygene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3,SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3(in all permutations and combinations including panels as discussedherein). In one embodiment, the subject is suffering from advancedadenomas or at risk for developing AJCC stage I, II, III or IV cancer.In another embodiment, the outcome is the survival of the subject aftera surgical resection, e.g. a noncurative or curative surgical resection.

Early detection of epigenetic silencing of one of more genes may providejustification for more definitive follow up of patients who havemolecular, but not yet all the pathological or clinical, featuresassociated with the malignancy. Identification of cancer at its earlieststage while it is still localized and readily treatable may improve theclinical outcome in patients.

Methods with a prognostic value should allow for the specific detectionof tumours and not detect (benign) adenomas, and thus provide for adifferential diagnosis between advanced adenoma versus benign adenoma.As shown in the detailed description (experimental part), gene promoterhypermethylation of at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein) was observed at a higher frequency in adenomas with concurrentcolorectal cancer when compared to adenomas from patients that did nothave colorectal cancer. This prognostic value is included within thedefinition of diagnosis.

In a related aspect, the invention provides a method for determining thehistopathological stage of cancer and in particular gastrointestinalcancer, such as colorectal cancer in a sample comprising detecting anepigenetic change in at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein), wherein detection of the epigenetic change is indicative of thehistopathological stage of the cancer, such as colorectal cancer forexample. All embodiments of the methods of the invention are herebyincorporated as appropriate and are not repeated for reasons ofconciseness. The epigenetic change is generally one causing genesilencing. Preferably, epigenetic silencing is detected by determinationof the methylation status and/or measurement of expression levels of theat least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) andthe methylation status and/or expression level of the gene or genes iscorrelated to a histopathological stage of cancer. In this method, asample is obtained from a subject suffering from, or suspected ofsuffering from any appropriate cancer in accordance with this invention,such as colorectal cancer for example. The methylation level of the atleast one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein), theexpression level of the at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein), or a combination thereof is determined and correlated to ahistopathological stage of the cancer. The “stage” of a cancer is adescriptor (usually numbers I to IV) of how much the cancer has spread.The stage often takes into account the size of a tumour, how deep it haspenetrated, whether it has invaded adjacent organs, if and how manylymph nodes it has metastasized to, and whether it has spread to distantorgans. Staging of cancer is important because the stage at diagnosis isthe biggest predictor of survival, and treatments are often changedbased on the stage. As aforementioned, the description of suitablemethods for determining epigenetic silencing of the at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) apply mutatismutandis to these aspects of the invention and are not repeated heresimply for reasons of conciseness.

In a specific embodiment, the invention provides a method fordetermining the histopathological stage of a gastrointestinal cancer,such as colorectal cancer and/or gastric cancer and/or oesophagealcancer or a predisposition thereto in a tissue or blood sample, orderivative thereof such as a plasma or serum sample. The most suitablegenes and combinations of genes are described hereinabove for thesespecific test samples (at least one gene selected from OSMR, SFRP1,NDRG4, GATA5, ADAM23, JPH3, SFRP2 and APC for blood samples for example)and are not repeated for reasons of conciseness.

In a further specific embodiment, the invention provides a method forpredicting or monitoring progression of an adenoma (to a carcinoma), inparticular in the context of gastrointestinal cancers such as colorectalcancer, comprising determining the methylation status of an NDRG2/NDRG4subfamily gene and in particular the NDRG4 gene in a suitable testsample, wherein an elevated or increased level of methylation indicatesthat the adenoma is more likely to progress to a carcinoma (than if thelevel of methylation is lower). This embodiment applies particularly tothe region of the NDRG4 gene which is amplified using primer set 1 asset out in table 2 above. Thus, in one embodiment, these methods employprimer set 1 in order to determine the methylation status of the NDRG4gene. As is discussed below, primer pair 1 allows distinguishing ofadenomas that progress to cancer from those that will not progress. Thisis highly important for cancer screening. The test sample may be anysuitable sample, as discussed extensively above. However, the sample isgenerally a suitable tissue sample, in particular an adenoma sample.

In a related embodiment, detecting increased levels of methylationtowards the transcription start site of the NDRG4 gene may also beuseful for monitoring the progression of cancer, and in particulargastrointestinal cancers such as colorectal cancer (CRC). As is shownherein, based upon the results obtained, it is predicted that spreadingof methylation from more 5′ regions of the promoter towards thetranscription start site correlates with cancer progression (for examplefrom adenoma to carcinoma). Thus, the invention provides a method forpredicting or monitoring progression of a gastrointestinal cancer, suchas CRC, comprising determining the methylation status of an NDRG2/NDRG4subfamily gene in a suitable test sample, wherein an elevated orincreased level of methylation towards the transcription start siteindicates that the cancer is more progressed than if the level ofmethylation is lower. The transcription start site and promoter sequenceare known from the published gene sequence information. Primer set 1 and2 as defined herein may be utilised as appropriate in these methods.Primer set 1 is used to determine the methylation status of the NDRG4gene closer to the transcription start site than primer set 2. Thus, acomparison of such results may be useful in these methods.

As stated herein the methods of the invention for diagnostic,prognostic, or personalised medicinal care are preferentially used inconnection with a gastrointestinal cancer, such as colorectal cancerand/or gastric cancer and/or oesophageal cancer. A number of techniquesare currently available for detection of colorectal cancer. Theseinclude:

-   -   Faecal occult blood tests (Guaiac and immunochemical)    -   Colonoscopy and/or sigmoidoscopy    -   X-ray after double-contrast barium enema or CT-colonography    -   Faecal DNA test (PreGen-Plus®)

More accurate screening, surveillance of higher-risk patients andimproved management of patient care may advantageously employ theseexisting methods and techniques to supplement the methods of theinvention.

Faecal DNA testing is an emerging technology in screening for colorectalcancer. Premalignant adenomas and cancers shed DNA markers from theircells which are not degraded during the digestive process and remainstable in the stool. Capture, followed by amplification, for exampleusing the Polymerase Chain Reaction, amplifies the DNA to detectablelevels for assay. The faecal DNA integrity assay has been proposed as auseful tool for the detection of colorectal cancer. The presence ofhigh-molecular-weight DNA fragments in stool is associated withcolorectal cancer and may be related to disease-associated differencesin the regulation of proliferation and apoptosis. Detecting colorectalcancer by testing stool for DNA may alternatively be based onidentifying oncogene mutations characteristic of colorectal neoplasiathat are detectable in exfoliated epithelial cells in the stool. Whileneoplastic bleeding is intermittent, epithelial shedding is continuous,potentially making stool-based DNA testing (also known as fecal DNA[f-DNA]) testing more sensitive than other methods. Commerciallyavailable stool-based DNA tests for colorectal cancer includePreGen-Plus™ (EXACT Sciences Corporation, Marlborough, Mass. 01752 USA)which is a single test that identifies the presence of 23 differentmicrosatellite (MSI) mutations known to be associated with CRC,including mutations in BAT-26. Additionally, 21 other point mutations inother genes associated with CRC are included in this test: adenomatouspolyposis coli (APC), K-ras, and protein and molecular size 53,000daltons (p53). This test is also designed to detect long DNA fragments,which have been specifically associated with cells called non-apoptoticcolonocytes, which are common in CRC.

Accordingly, molecular screening of faecal samples focused on oncogenemutations and/or DNA integrity may complement the methods of presentinvention. In specific embodiments, the methods of the invention areused in combination with detecting DNA integrity, or at least one DNAoncogene mutation, or a combination of both detecting DNA integrity andat least one DNA oncogene mutation in the sample in order to detect apredisposition to, or the incidence of, colorectal cancer. The methodsmay be carried out on a faecal sample. In one embodiment the method mayalso include the step of obtaining and/or processing the sample.

Testing can be performed diagnostically or in conjunction with atherapeutic regimen. Epigenetic loss of function of at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) can be rescued by theuse of DNA demethylating agents and/or DNA methyltransferase inhibitors.Testing can be used to determine what therapeutic or preventive regimento employ on a patient and be used to monitor efficacy of a therapeuticregimen.

Accordingly, the invention also provides a method for predicting thelikelihood of successful treatment of a cancer as defined herein and inparticular gastrointestinal cancer, such as colorectal cancer and/orgastric cancer and/or oesophageal cancer with a DNA demethylating agentand/or a DNA methyltransferase inhibitor and/or HDAC inhibitorcomprising detecting an epigenetic change in at least one gene selectedfrom an NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR,GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations and combinationsincluding panels as discussed herein), wherein detection of theepigenetic change is indicative that the likelihood of successfultreatment is higher than if the epigenetic modification is not detected.Alternatively, the method comprises measurement of expression levels ofthe at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein),wherein a reduced level of expression indicates the likelihood ofsuccessful treatment of cancer is higher than if the at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) is expressed at ahigher level. For the avoidance of doubt it is stated that thedescription of suitable methods (sample types, cancer types, panels ofgenes etc.) for determining epigenetic silencing of the at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) apply mutatismutandis to these aspects of the invention and are not repeated heresimply for reasons of conciseness.

In an opposite scenario, the invention provides a method for predictingthe likelihood of resistance to treatment of colorectal cancer with aDNA demethylating agent and/or DNA methyltransferase inhibitor and/orHDAC inhibitor comprising detecting an epigenetic change in at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein), wherein detection ofthe epigenetic change is indicative that the likelihood of resistance totreatment is lower than if the epigenetic modification is not detected.

Alternatively, the method comprises measurement of expression levels ofthe at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein),wherein a higher level of expression indicates the likelihood ofresistance to treatment of cancer is higher than if the at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) is expressed at areduced level.

Thus, the patient population may be selected for treatment on the basisof their methylation status with respect to the relevant at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein—such as where the atleast one gene is selected from GATA4, OSMR, NDRG4 and SFRP2 or selectedfrom OSMR, NDRG4, GATA5 and ADAM23 where tissues or bodily fluid and inparticular faecal or blood based samples and in particular plasmasamples are utilised), which leads to down regulation of gene expressionof the corresponding gene. This leads to a much more focussed andpersonalised form of medicine and thus leads to improved success ratessince patients will be treated with drugs which are most likely to beeffective. The description of suitable methods for determiningepigenetic silencing of the at least one gene selected from GATA4, OSMR,NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 apply mutatis mutandis to theseaspects of the invention and are not repeated here simply for reasons ofconciseness.

In certain aspects, epigenetic loss of function of at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein) in adenoma canidentify the need for treatment. Subjects having a disease such as colonneoplasia may be assayed for methylation of at least one gene selectedfrom an NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR,GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations and combinationsincluding panels as discussed herein). Alternatively, the subject may beundergoing routine screening and may not necessarily be suspected ofhaving a disease such as colon neoplasia. Detecting methylation of atleast one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) inan adenoma can be used to improve sensitivity and/or specificity fordetecting a colon neoplasia, since such advanced adenoma may indicatethat the probable course of the adenoma is development to a carcinoma.In such case, preventive treatment may be recommended and involveresection of the adenoma.

Accordingly, the invention provides a method for predicting suitabletreatment of an adenoma obtained from a subject, comprising determiningthe methylation status of at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein) in an adenoma, wherein if the at least one gene selected from anNDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) is methylated, in particularhypermethylated, the need for treatment of the adenoma is identified.Preferably, the treatment comprises resection of the adenoma.

In an opposite scenario, the invention provides a method for predictingsuitable treatment of an adenoma obtained from a subject, comprisingdetermining the methylation status of at least one gene selected from atleast one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein) inan adenoma, wherein if the at least one gene selected from anNDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) is unmethylated or methylated to a lesserdegree, it is decided that there is no need of resection of the adenoma.The description of suitable methods for determining epigenetic silencingof the at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein)apply mutatis mutandis to these aspects of the invention and are notrepeated here simply for reasons of conciseness. The adenomas aretypically of colonic origin in certain embodiments.

The invention further provides for a method of selecting a suitabletreatment regimen for cancer or predisposition to cancer comprisingdetermining epigenetic silencing of a NDRG4/2 family gene in a sampleobtained from a subject, wherein if the gene is epigenetically silenced,in particular hypermethylated or reduced expressed, a DNA demethylatingagent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitoris selected for treatment.

In an opposite scenario, the invention provides for a method ofselecting a suitable treatment regimen for cancer or predisposition tocancer comprising determining the methylation status and/or expressionlevel of a NDRG4/2 family gene in a sample obtained from a subject,wherein if the gene is unmethylated or higher expressed, treatment witha DNA demethylating agent and/or a DNA methyltransferase inhibitorand/or a HDAC inhibitor is contra-indicated. Thus, alternative treatmentshould be explored.

In a related aspect, the invention also provides a method of selecting asuitable treatment regimen for cancer, in particular a gastrointestinalcancer such as colorectal cancer (as defined herein), comprisingdetecting an epigenetic change in at least one gene selected from anNDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein), wherein detection of the epigenetic changeresults in selection of a DNA demethylating agent and/or a DNAmethyltransferase inhibitor and/or a HDAC inhibitor for treatment andwherein if the epigenetic change is not detected, a DNA demethylatingagent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitoris not selected for treatment. In the event that the epigenetic changeis not detected (for example through gene expression detection or anyother suitable method), alternative treatments should be explored. Thedescription of suitable methods for determining epigenetic silencing ofthe at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein)apply mutatis mutandis to these aspects of the invention and are notrepeated here simply for reasons of conciseness. In embodiments whereblood and in particular plasma or serum samples are utilised, the atleast one gene may be selected from OSMR, SFRP1, NDRG4, GATA5, ADAM23,JPH3, SFRP2 and APC. Suitable panels in this context comprise, consistessentially of or consist of OSMR, NDRG4, GATA5 and ADAM23. Additionallyor alternatively, the at least one gene may be selected from TFPI2,BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3, such as from TFPI2, BNIP3,FOXE1, SYNE1 and SOX17, in particular TFPI2.

In embodiments where faecal samples are employed, the at least one genemay be selected from GATA4, OSMR, NDRG4, GATA5, SERP1, ADAM23, JPH3,SFRP2, APC and MGMT. In addition, or alternatively, the at least onegene may be selected from TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3,and JAM3, such as from TFPI2, FOXE1, SYNE1, SOZ17, PHACTR3 and JAM3, inparticular TFPI2. Suitable panels, as defined herein, are alsoenvisaged, such as a panel comprising, consisting essentially of orconsisting of OSMR, NDRG4, GATA4 and SFRP2 for example.

In embodiments in which tissue samples are utilised, the methods maycomprise, consist essentially of or consist of detecting an epigeneticchange in a panel of genes comprising OSMR, GATA4 and ADAM23 or OSMR,GATA4 and GATA5. The tissue sample may comprise, consist essentially ofor consist of a colon and/or rectal and/or appendix sample.

In another aspect, the invention provides for a method of treatingcancer and in particular colorectal cancer in a subject comprisingadministration of a DNA demethylating agent and/or a HDAC inhibitorand/or a DNA methyltransferase inhibitor wherein the subject has beenselected for treatment on the basis of a method of the invention.Accordingly, the description of suitable methods for determiningepigenetic silencing of the at least one gene selected from anNDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) apply mutatis mutandis to these aspects ofthe invention and are not repeated here simply for reasons ofconciseness. Thus, in embodiments where blood and in particular plasmaor serum samples are utilised, the at least one gene may be selectedfrom OSMR, SFRP1, NDRG4, GATA5, ADAM23, JPH3, SFRP2 and APC. Suitablepanels in this context comprise, consist essentially of or consist ofOSMR, NDRG4, GATA5 and ADAM23.

Additionally or alternatively, the at least one gene may be selectedfrom TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3, such as fromTFPI2, BNIP3, FOXE1, SYNE1 and SOX17, in particular TFPI2.

In embodiments where faecal samples are employed, the at least one genemay be selected from GATA4, OSMR, NDRG4, GATA5, SERP1, ADAM23, JPH3,SFRP2, APC and MGMT. In addition, or alternatively, the at least onegene may be selected from TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3,and JAM3, such as from TFPI2, FOXE1, SYNE1, SOZ17, PHACTR3 and JAM3, inparticular TFPI2. Suitable panels, as defined herein, are alsoenvisaged, such as a panel comprising, consisting essentially of orconsisting of OSMR, NDRG4, GATA4 and SFRP2 for example.

In embodiments in which tissue samples are utilised, the methods maycomprise, consist essentially of or consist of detecting an epigeneticchange in a panel of genes comprising OSMR, GATA4 and ADAM23 or OSMR,GATA4 and GATA5. The tissue sample may comprise, consist essentially ofor consist of a colon and/or rectal and/or appendix sample.

Thus, for the patient population where the at least one gene selectedfrom an NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR,GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations and combinationsincluding panels as discussed herein) is methylated, which leads todecreased gene expression, this type of treatment is recommended. Thismethod is referred to hereinafter as the “method of treatment” aspect ofthe invention.

In a related aspect, the invention also provides for the use of a DNAdemethylating agent and/or a DNA methyltransferase inhibitor and/or HDACinhibitor (in the manufacture of a medicament for use) in treatingcancer, and in particular a gastrointestinal cancer, such as colorectalcancer and/or gastric cancer and/or oesophageal cancer in a subject,wherein the subject has been selected for treatment on the basis of themethods of the invention. Likewise, the invention provides a DNAdemethylating agent and/or a DNA methyltransferase inhibitor and/or HDACinhibitor for use in treating cancer, and in particular agastrointestinal cancer, such as colorectal cancer and/or gastric cancerand/or oesophageal cancer in a subject, wherein the subject has beenselected for treatment on the basis of the methods of the invention.

For all of the relevant methods (pharmacogenetic methods, treatmentregimen methods and methods of treatment) of the invention, the DNAdemethylating agent may be any agent capable of up regulatingtranscription of at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein—such as at least one gene selected from GATA4, OSMR, NDRG4, GATA5and ADAM23). A preferred DNA demethylating agent comprises, consistsessentially of or consists of a DNA methyltransferase inhibitor. The DNAmethyltransferase inhibitor may be any suitable inhibitor of DNAmethyltransferase which is suitable for treating cancer in the presenceof methylation of the at least one gene selected from an NDRG2/NDRG4subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23,JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3 (in all permutations and combinations including panels as discussedherein—such as at least one gene selected from OSMR, NDRG4, GATA5 andADAM23). As is shown in the experimental section below, methylation ofthe at least one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein suchas at least one gene selected from OSMR, NDRG4, GATA5 and ADAM23) islinked to colorectal cancer and so preventing this methylation ispredicted to help to treat a gastrointestinal cancer, such as colorectalcancer and/or gastric cancer and/or oesophageal cancer.

The DNA methyltransferase inhibitor may, in one embodiment, be one whichreduces expression of DNMT genes, such as suitable antisense molecules,or siRNA molecules which mediate RNAi for example. The design of asuitable siRNA molecule is within the capability of the skilled personand suitable molecules can be made to order by commercial entities (seefor example, www.ambion.com). In embodiments, the DNA methyltransferasegene is (human) DNMT1.

Alternatively, the agent may be a direct inhibitor of DNMTs. Examplesinclude modified nucleotides such as phosphorothioate modifiedoligonucleotides (FIG. 6 of Villar-Garea, A. And Esteller, M. DNAdemethylating agents and chromatin-remodelling drugs: which, how andwhy? Current Drug Metabolism, 2003, 4, 11-31) and nucleosides andnucleotides such as cytidine analogues. Suitable examples of cytidineanalogues include 5-azacytidine, 5-aza-2′-deoxycytidine,5-fluouro-2′-deoxycytidine, pseudoisocytidine,5,6-dihydro-5-azacytidine, 1-β-D-arabinofuranosyl-5-azacytosine (knownas fazabarine) (see FIG. 4 of Villar-Garea, A. And Esteller, M. DNAdemethylating agents and chromatin-remodelling drugs: which, how andwhy? Current Drug Metabolism, 2003, 4, 11-31).

In another embodiment, the DNA methyltransferase inhibitor comprisesDecitabine. Full details of this drug can be found at www.supergen.comfor example.

Additional DNMT inhibitors include S-Adenosyl-Methionine (SAM) relatedcompounds like ethyl group donors such as L-ethionine and non-alkylatingagents such as S-adenosyl-homocysteine (SAH), sinefungin,(S)-6-methyl-6-deaminosine fungin, 6-deaminosinefungin,N4-adenosyl-N4-methyl-2,4-diaminobutanoic acid,5′-methylthio-5′-deoxyadenosine (MTA) and 5′-amino-5′-deoxyadenosine(Villar-Garea, A. And Esteller, M. DNA demethylating agents andchromatin-remodelling drugs: which, how and why? Current DrugMetabolism, 2003, 4, 11-31).

Further agents which may alter DNA methylation and which may, therefore,be useful in the present compositions include organohalogenatedcompounds such as chloroform etc, procianamide, intercalating agentssuch as mitomycin C, 4-aminobiphenyl etc, inorganic salts of arsenic andselenium and antibiotics such as kanamycin, hygromycin and cefotaxim(Villar-Garea, A. And Esteller, M. DNA demethylating agents andchromatin-remodelling drugs: which, how and why? Current DrugMetabolism, 2003, 4, 11-31).

Useful DNMT inhibitors in the present invention comprise, consistsessentially of or consists of 5-azacytidine and/or zebulaine.

As discussed above, one challenge faced by researchers investigatingcolorectal cancer is the diversity of DNA present in stool samples. TheDNA of interest represents only a very small percentage of the total DNAisolated from stool. Therefore, along with the exploration of suitableDNA markers, techniques for improved DNA isolation and enrichment of thehuman DNA component from faecal samples are required for more sensitivecancer detection.

Most techniques for improved sensitivity of cancer detection from faecalsamples focus on improvements in recovery of target human DNA from thetotal DNA. The inventors have successfully improved the sensitivity ofdetection of colorectal cancer in faecal samples by increasing theamount of DNA used in the detection reactions. Increasing the amount ofDNA in the detection reaction goes along with an increase in substancesco-purified with the DNA.

An increase in the amount of impurities may be expected to result inPCR-inhibition, and therefore an increased level of input DNA in thedetection reaction has not been previously explored for improving thesensitivity of cancer detection in faecal samples.

Accordingly, in a further aspect, the invention provides a method ofprocessing a faecal sample to isolate and prepare DNA for use indetecting a predisposition to, or the incidence of, colorectal cancer ina faecal sample comprising:

(a) isolating DNA from the faecal sample

(b) subjecting at least 2.5 μg of the isolated DNA per amplificationreaction required to treatment with a reagent which selectively modifiesunmethylated cytosine residues in the DNA contained in the sample toproduce detectable modified residues but which does not modifymethylated cytosine residues(c) amplifying the treated isolated DNA.

Thus, the inventors have found that by including at least 2.5 g ofisolated DNA in the reagent treatment step for every downstreamamplification that is required, improved detection methods using stoolsamples are achieved. The amount of DNA is expressed per amplificationreaction required in particular to allow for multiple parallel reactionsto be carried out on the same sample. For example, test and controlsamples can then be run in parallel. Also, where detection of anepigenetic change, preferably methylation, in at least one gene selectedfrom an NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR,GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations and combinationsincluding panels as discussed herein) is not carried out in the samereaction (by use of appropriate fluorophores for example) each of apanel of genes may be assessed in a separate reaction. Thus a singlestarting sample may need to be split into a plurality of sub-samples, asrequired. This improves the accuracy of the results obtained byminimizing inter-sample variations. The amount of isolated DNA peramplification reaction is at least approximately 2.5 μg, 3 μg, 4 μg, 5μg, 7.5 μg, 10 μg etc. to improve sensitivity, and is most preferablyapproximately 2.5 μg.

In one embodiment, the method further comprises, preferably prior toisolation of DNA from the sample, adding a homogenization buffer to thefaecal sample. Any suitable buffer may be utilized. Useful buffers arecommercially available, for example from Amresco.

The reagent which selectively modifies unmethylated cytosine residues inthe DNA contained in the sample to produce detectable modified residuesbut which does not modify methylated cytosine residues the reagentpreferably comprises, consists essentially of or consists of abisulphite reagent. Suitable reagents are discussed herein, whichdiscussion applies mutatis mutandis. In specific embodiments, thebisulphite reagent comprises, consists essentially of or consists ofsodium bisulphite.

In a specific embodiment, between treatment of the isolated DNA with thereagent and amplification of the treated isolated DNA, the treatedisolated DNA is concentrated. Any suitable DNA concentration method maybe utilised. For example, a DNA-binding reagent may be utilised in orderto concentrate DNA from the sample. DNA-binding reagents may be selectedfrom DNA-binding buffers, DNA-binding filters, DNA-binding columns etc.and may require use of a centrifugation step. Suitable kits arecommercially available, such as the ZYMO Clean and Concentrator Kitavailable from Zymo Research.

In order to achieve the necessary recovery of DNA from the faecalsample, the faecal sample may be at least approximately 4 g in weight.The faecal sample may be anywhere between approximately 2 g and 10 g inweight and is most preferably around 4 g in weight.

The methods of the invention may thus include steps such as:

-   -   obtaining and processing a stool sample from the subject under        test, wherein preferably around (at least) 4 g stool is obtained    -   adding homogenization buffer, preferably directly after        defecation (the subject may add this themselves). The buffer may        be added at any suitable ratio, such as at 1:7 for example    -   isolating DNA from the stool sample. As mentioned above, any        suitable DNA isolation technique may be employed. This may        involve (a double) low speed centrifugation. Isolation may        require RNase A and Proteinase K treatment followed by DNA        extraction, for example by phenol/chloroform extraction. Other        DNA purification techniques can be used, as discussed herein    -   subjecting the obtained DNA to bisulphite conversion, such as        subjecting at least 18 to 32 μg DNA to bisulphite conversion or        subjecting at least 2.5 μg DNA to bisulphite conversion for each        PCR reaction to be done (input expressed per PCR reaction for        reason of multiplexing as discussed earlier)    -   concentrating the amount of bisulphite treated DNA obtained from        the at least 18 to 32 μg untreated DNA    -   amplifying the amount of bisulphite converted DNA. The amount to        be amplified may be equivalent to 10 to 2.5 μg unconverted DNA        (this equals the amounts for 4 marker panels down to use of a        single marker).

The sensitivity of the methods for processing a faecal sample may beimproved further by combining them with known methods for isolating DNAfrom a faecal sample. For example, the human DNA component may bepurified from a stool sample using streptavidin-bound magnetic beads(Dong et al., 2001; Ahlquist et al., 2000). In a further embodiment, anelectrophoresis-driven separation of target DNA sequences, usingoligonucleotide capture probes immobilized in an acrylamide gel (Whitneyet al., 2004) may be utilised in order to purify human DNA from thestool sample. In a still further embodiment, which may be used in thealternative or in combination with earlier embodiments, faecal samplesmay be frozen as quickly as possible after collection in order topreserve DNA integrity. As discussed above, DNA integrity may also beusefully tested in terms of diagnosing colorectal cancer. Additionallyor alternatively, stabilization buffer may be added to the faecalsamples before transport of the samples (Olson et al., 2005). In a yetfurther complementary embodiment, Methyl-binding domain (MBD) proteinmay be utilised to enrich methylated human DNA from a faecal sample, inorder to specifically improve sensitivity for detecting methylated DNAmarkers in the sample (Zou et al., Clin Chem. 2007 September;53(9):1646-51).

In specific aspects, the methods of processing a faecal sample accordingto the invention are combined with the other methods of the invention inorder to provide improved diagnosis, histopathological analysis,pharmaogenomic analysis etc. of colorectal cancer. Accordingly, allembodiments of the methods of the invention apply mutatis mutandis Thus,the methods of the invention can be performed on the amplified treatedDNA to provide particularly sensitive methods relating to colorectalcancer for example.

In a still further aspect, the invention provides a method ofdetermining the methylation status of at least one gene in a bloodsample, in particular a blood plasma or serum sample, comprising:

(a) isolating DNA from a blood plasma or serum sample

(b) subjecting the isolated DNA to treatment with a reagent whichselectively modifies unmethylated cytosine residues in the DNA containedin the sample to produce detectable modified residues but which does notmodify methylated cytosine residues

(c) amplifying the treated isolated DNA in order to determine themethylation status of at least one gene, characterised in that 0.07 to0.72 ml blood plasma or serum sample equivalent of DNA is used peramplification reaction.

The methods thus utilise small volumes in the amplification reactionsyet still maintain high sensitivity and specificity of detection. Thus,as discussed herein, a single blood sample may be advantageouslyutilised to determine the methylation status of a panel of genes in oneembodiment. The volumes may be anywhere between around 0.07 and around0.72 ml blood plasma or serum equivalent, and as discussed belowpreferably plasma equivalent, of DNA per amplification reaction. Inspecific embodiments, between around 0.07, 0.10, 0.15 and 0.50, 0.60,0.70 ml, such as between 0.07 and 0.15, 0.16, 0.17, 0.18 or 0.19 mlblood plasma or serum equivalent of DNA is used per amplificationreaction. In a specific embodiment, substantially the same selectedvolumes of blood plasma or serum sample, equivalent of DNA is used foreach amplification reaction carried out. Thus, where multipleamplifications are carried out based upon a single blood sample takenfrom a subject, each amplification will utilise 0.07 to 0.72 ml bloodplasma or serum sample, equivalent of DNA.

The blood plasma or serum sample may be derived from whole blood or anysuitable plasma or serum containing parts/fractions thereof asappropriate. In specific embodiments, the blood plasma or serum samplecomprises, consist essentially of or consists of plasma. The bloodsample, from which the plasma or serum is derived may be collected usingany suitable method. Many such methods are well known in the art. In oneembodiment, the methods of the invention also incorporate the step ofobtaining the blood sample and/or the plasma or serum sample from wholeblood. Any appropriate blood sample may be utilised in the methods ofthe invention, provided it contains sufficient (free floating) DNA. In aspecific embodiment, the volume of the blood sample, or derivativethereof that is utilised in the methods is around 5 to 15 ml, such as 10ml.

Blood samples, or derivatives thereof and in particular plasma or serumsamples, may be stored prior to use in the methods of the invention onceobtained. They may be frozen, for example, at a suitable temperature.Suitable temperatures may be between around 0° C., −1° C., −2° C., −3°C., −4° C. and −20° C., −30° C., −40° C., −50° C., −60° C., −70° C.,−80° C., −90° C. etc., such as around −80° C. They may also be stored atother temperatures, such as at 4° C. or at room temperature dependingupon their form. In one specific embodiment, plasma or serum is dried toallow storage at non-freezing temperatures. The drying may compriselyophilization for example, although other dehydration techniques may beemployed. Where plasma or serum is stored at temperatures greater (i.e.warmer) than freezing, and in particular greater than −80° C.,antimicrobial agents such as antibiotics may be added to the sample toprevent spoiling.

In one embodiment, stabilizers are added to the blood sample, orderivative thereof, in particular serum or plasma. This is particularlyrelevant where the sample is not frozen. In one specific embodiment,where the sample is serum, stabilizers such as stabilizers selected fromEDTA and/or citrate and/or heparin are employed. In a furtherembodiment, where the sample is plasma, stabilizers such as stabilizersselected from citrate and/or heparin may be utilised.

It is preferred that the blood plasma or serum sample comprises,consists essentially of or consists of a plasma sample. Plasma may bederived from whole blood by any suitable means. In one embodiment, theplasma sample is obtained by centrifugation of whole blood.Centrifugation may be carried out at any suitable speed and for anysuitable period of time and under any suitable conditions as may bedetermined by one skilled in the art. For example, centrifugation may becarried out at between around 1000 and 3000 g. Centrifugation may becarried out for between around 1, 2, 3, 4, or 5 and 10, 11, 12, 13, 14or 15 minutes for example. Centrifugation may be carried out at lowtemperatures, such as between around 0 and 5° C., for example 4° C., tomaintain integrity of the sample. Multiple centrifugation steps may beemployed in order to obtain the plasma sample. In a specific embodiment,two centrifugation steps are employed to obtain the plasma sample.

It has been shown that sensitivity of the methods of the invention maybe improved by excluding samples with a plasma (or serum) volume lessthan around 1 to 3 ml and in particular around 2 ml (such as 1.5 to 2.5ml) prior to isolating DNA. Thus, the methods may comprise determiningthe volume of plasma (or serum) obtained from a blood sample prior toDNA isolation. If the volume of the plasma (or serum) obtained from theblood sample is less than around 1 to 3 ml and in particular around 2 ml(such as 1.5 to 2.5 ml), the sample is excluded from further assessment.

As stated herein, the methods are useful for determining the methylationstatus of at least one gene. By “determining the methylation status” ismeant determining the presence or absence of 5-methylcytosine (“5-mCyt”)at one or a plurality of (functionally relevant) CpG dinucleotideswithin the DNA sequence of the at least one gene. In particular,aberrant methylation, which may be referred to as hypermethylation, ofthe at least one gene may be detected. Typically, the methylation statusis determined in one or more CpG islands in the at least one gene. TheseCpG islands are often found in the promoter region of the gene(s). Thus,CpG dinucleotides are typically concentrated in the promoter regions andexons of human genes and the methylation status of these CpG residues isof functional importance to whether the at least one gene is expressed.Since CpG dinucleotides susceptible to methylation are typicallyconcentrated in the promoter region, exons and introns of human genes,promoter, exon and intron regions may be assessed in order to determinethe methylation status of the at least one gene. A “promoter” is aregion extending typically between approximately 1 Kb, 500 bp or 150 to300 bp upstream from the transcription start site. The CpG island maysurround or be positioned around the transcription start site of the atleast one gene.

The methods of the invention involve isolating/extracting/purifying DNAfrom the blood plasma or serum sample. Any suitable DNA isolationtechnique may be utilised, as discussed herein, which discussion applieshere mutatis mutandis. Likewise, suitable methods and kits for isolatingDNA from blood samples which are commercially available are discussedand exemplified herein, which discussion applies here mutatis mutandis(see table 1). Thus, as can be derived from the table, DNA isolation maybe carried out using silica-membranes, isopropanol or magnetic beadbased methods for example.

The methods of the invention may also, as appropriate, incorporatequantification of isolated/extracted/purified DNA in the sample.Quantification of the DNA in the sample may be achieved using anysuitable means. Quantitation of nucleic acids may, for example, be basedupon use of a spectrophotometer, a fluorometer or a UV transilluminator.Examples of suitable techniques are described in standard texts such asMolecular Cloning—A Laboratory Manual (Third Edition), Sambrook andRussell (see in particular Appendix 8 therein). In one embodiment, kitssuch as the Picogreen® dsDNA quantitation kit available from MolecularProbes, Invitrogen may be employed to quantify the DNA.

The methods of this aspect of the invention (and other aspects of theinvention which involve certain types of methylation detection) relyupon a reagent which selectively modifies unmethylated cytosine residuesin the DNA contained in the sample to produce detectable modifiedresidues but which does not modify methylated cytosine residues. Anysuitable reagent may be utilised in the methods of the invention.Examples include bisulphite, hydrogen sulphite and disulphite reagentsand suitable mixtures thereof. In an embodiment of the invention, thereagent comprises, consists essentially of or consists of a bisulphitereagent. In particular, the reagent may comprise, consist essentially ofor consist of sodium bisulphite.

In a specific embodiment, following treatment of the isolated DNA withthe reagent, and preferably between treatment of the isolated DNA withthe reagent and amplification of the treated isolated DNA, the treatedisolated DNA is concentrated. Any suitable DNA concentration method maybe utilised. For example, a DNA-binding reagent may be utilised in orderto concentrate DNA from the sample. DNA-binding reagents may be selectedfrom DNA-binding buffers, DNA-binding filters, DNA-binding columns etc.and may require use of a centrifugation step. Suitable kits arecommercially available, such as the ZYMO Clean and Concentrator Kitavailable from Zymo Research.

In one specific embodiment, the at least one gene whose methylationstatus is determined is selected from OSMR, SFRP1, NDRG4, GATA5, ADAM23,JPH3, SFRP2 and APC. As is discussed in detail herein, the methylationstatus of these genes in blood plasma or serum samples is correlatedwith the incidence of cancer and in particular colorectal cancer.Details of these genes are provided herein which discussion applies tothis aspect mutatis mutandis.

In a specific embodiment, the at least one gene is selected from OSMR,NDRG4, GATA5 and ADAM23 since these four genes have been shown to beparticularly reliably linked to the incidence of colorectal cancer usingblood derived samples, in particular plasma sample.

Also, these genes have been shown to be linked to early stage colorectalcancer. Accordingly, the invention provides a method of determining themethylation status of at least one gene selected from OSMR, NDRG4, GATA5and ADAM23 in a blood sample, in particular a blood plasma or serumsample, comprising:

(a) isolating DNA from a blood plasma or serum sample

(b) subjecting the isolated DNA to treatment with a reagent whichselectively modifies unmethylated cytosine residues in the DNA containedin the sample to produce detectable modified residues but which does notmodify methylated cytosine residues

(c) amplifying the treated isolated DNA in order to determine themethylation status of at least one gene, characterised in that 0.07 to0.72 ml blood plasma or serum sample equivalent of DNA is used peramplification reaction.

This method may be utilised in order to diagnose early stage colorectalcancer, in particular stage 0 to II colorectal cancer. It may also beused to stage colorectal cancer—detection of methylated gene or genesindicates an early stage of cancer. Corresponding methods and kits arealso envisaged. These methods may additionally or alternatively beusefully applied applied to determine the methylation status of at leastone gene selected from TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 andJAM3, such as from TFPI2, BNIP3, FOXE1, STNE1 and SOX17, in particularTFPI2.

Moreover, in order to improve the sensitivity of the methods of theinvention the methods may comprise determining the methylation status ofa panel of genes comprising at least two, three, four, five or six (ofthe) genes. Thus, in one embodiment, the at least one gene forms part ofa panel of genes comprising at least two, three, four, five or sixgenes, wherein the methylation status of each of the genes isdetermined. The panel of genes may comprise, consist essentially of orconsist of two, three, four, five or six genes. Suitable panels arediscussed herein in respect of other aspects of the invention. Thatdiscussion and those embodiments apply here mutatis mutandis.

In specific embodiments, the panel of genes comprises, consistsessentially of or consists of OSMR, NDRG4, GATA5 and ADAM23. This panelmay be useful in the diagnosis of early stage colorectal cancer, such asstage 0 to II colorectal cancer.

It is noted that for each gene, it may be possible to determine themethylation status of the gene, in a plurality of locations within thesame gene (as discussed herein). Thus, for example, a gene mayincorporate more than one CpG island, or multiple sites within the sameCpG island may be investigated as appropriate.

As discussed in greater detail herein, the determination of themethylation status of each of the panel of genes may be carried out in asingle reaction. Many suitable techniques allowing multiplexing areavailable and may be utilised in the present invention. Most depend uponuse of suitable fluorescent molecules having distinguishable emissionspectra. The skilled person can readily select from the manyfluorophores available to determine which can be used in a multiplexingcontext.

In one embodiment, a universal quencher is utilised together withsuitable fluorophore donors each having a distinguishable emissionwavelength maximum. One suitable quencher is DABCYL. Together with asuitable quencher such as DABCYL the following fluorophores may each beutilised to allow multiplexing: Coumarin (emission maximum of 475 nm),EDANS (491 nm), fluorescein (515 nm), Lucifer yellow (523 nm), BODIPY(525 nm), Eosine (543 nm), tetramethylrhodamine (575 nm) and texas red(615 nm) (Tyagi et al., Nature Biotechnology, Vol. 16, Jan. 1998;49-53).

As discussed above, the methylation status of additional genes may alsobe determined in order to supplement the methods of the invention. Othergenes involved in the establishment of colorectal cancer may be selectedfrom the group consisting of CHFR, MGMT, p16, Vimentin, p14, RASSF1a,RAB32, SEPTIN-9, RASSF2A, ALX4 and SMARCA3.

The final step of the methods of the invention involve amplifying thetreated isolated DNA in order to determine the methylation status of atleast one gene. As discussed above, this amplification utilises 0.07 to0.72 ml blood plasma or serum sample, equivalent of DNA peramplification reaction. Any suitable amplification technique may beutilised. In a specific embodiment, the amplifying step comprises,consists essentially of or consists of the polymerase chain reaction(PCR). It should be noted that whilst PCR is a preferred amplificationmethod, to include variants on the basic technique such as nested PCR,equivalents may also be included within the scope of the invention.Examples include without limitation isothermal amplification techniquessuch as NASBA, 3SR, TMA and triamplification, all of which are wellknown in the art and commercially available. Other suitableamplification methods without limitation include the ligase chainreaction (LCR) (Barringer et al, 1990), MLPA, selective amplification oftarget polynucleotide sequences (U.S. Pat. No. 6,410,276), consensussequence primed polymerase chain reaction (U.S. Pat. No. 4,437,975),invader technology (Third Wave Technologies, Madison, Wis.), stranddisplacement technology, arbitrarily primed polymerase chain reaction(WO90/06995) and nick displacement amplification (WO2004/067726).

Various amplification based assays for determining the methylationstatus of at least one gene are known in the art, and can be used inconjunction with the present invention. These assays (includingtechniques such as methylation specific PCR) are described in greaterdetail herein, which description applies here mutatis mutandis and isnot repeated simply for reasons of conciseness.

In specific embodiments, the methods of the invention employ or relyupon or utilise primers and/or probes selected from the primers andprobes comprising the nucleotide sequences set forth in the relevanttables above (such as tables 2 to 18 and in particular tables 4 to 10)to determine the methylation status of the at least one gene. The tablespresent specific primer and probe combinations for certain preferredgenes whose methylation status may be determined according to themethods of the invention.

Sequence variation that reflects the methylation status at CpGdinucleotides in the original genomic DNA offers two approaches toprimer design. Both primer types may be utilised in the methods of theinvention as discussed in detail herein, which discussion appliesmutatis mutandis here. Suitable probes may also be employed, asdescribed herein.

When determining methylation status, it may be beneficial to includesuitable controls in order to ensure the method chosen to assess thisparameter is working correctly and reliably. Suitable (positive andnegative) controls are discussed in detail herein, which discussionapplies mutatis mutandis.

As can be derived from the discussion and examples herein, themethylation status of the at least one gene may be correlated with theincidence of a disease for specific genes and specific diseases.Accordingly, the methods of the invention may be used in order to detecta predisposition to, or the incidence of, any disease for which genemethylation plays a role. In a specific embodiment, the diseasecomprises a cell proliferative disorder, although in principle anydisease may be diagnosed according to these methods provided that genemethylation can be determined in an appropriate blood plasma or serumsample. The cell proliferative disorder may comprise, consistessentially of or consist of cancer for example. In particular, thecancer may comprise, consist essentially of or consist of agastrointestinal cancer, such as colorectal cancer and/or gastric cancerand/or oesophageal cancer and in particular colorectal cancer forexample. Further specific gastrointestinal cancers are discussed aboveand each may be applicable to the present methods. As discussed herein,the methods may have particular application to early stage colorectalcancer, such as stage 0 to II colorectal cancer.

The invention also provides kits which may be used in order to carry outthe methods of the invention. The kits may incorporate any of thevarious features, aspects and embodiments mentioned in connection withthe various methods (and uses) of the invention above.

Thus, a kit is provided for:

-   -   (a) predicting the likelihood of successful treatment of cancer        (as defined herein) and in particular a gastrointestinal cancer,        such as colorectal cancer and/or gastric cancer and/or        oesophageal cancer and/or the likelihood of resistance to        treatment of cancer and in particular a gastrointestinal cancer,        such as colorectal cancer and/or gastric cancer and/or        oesophageal cancer with a DNA damaging agent and/or a DNA        methyltransferase inhibitor and/or a HDAC inhibitor, and/or    -   (b) selecting a suitable treatment regimen for cancer and in        particular a gastrointestinal cancer, such as colorectal cancer        and/or gastric cancer and/or oesophageal cancer and/or    -   (c) diagnosing cancer and in particular a gastrointestinal        cancer, such as colorectal cancer and/or gastric cancer and/or        oesophageal cancer or a predisposition thereto, and/or    -   (d) determining the histopathological stage of cancer and in        particular a gastrointestinal cancer, such as colorectal cancer        and/or gastric cancer and/or oesophageal cancer or a        predisposition thereto in a sample comprising carrier means        containing therein a set of primers for use in detecting the        methylation status of at least one gene selected from an        NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR,        GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,        FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations and        combinations including panels as discussed herein, in particular        with respect to the methods of the invention). For example in        one specific embodiment, the kit comprises carrier means        containing therein a set of primers for use in detecting the        methylation status of at least one gene selected from OSMR,        SFRP1, NDRG4, GATA5, ADAM23, JPH3, SFRP2 and APC. Any of the        NDRG2/NDRG4-family genes may be assessed using the kits of the        invention. A more detailed discussion of family members is        provided above.

Thus, the kit may include suitable primers for determining whether theNDRG2/NDRG4-family gene and preferably the NDRG4 and/or NDRG2 gene ismethylated. These primers may comprise any of the primers discussed indetail in respect of the various methods of the invention which may beemployed in order to determine the methylation status of theNDRG2/NDRG4-family gene and preferably the NDRG4 and/or NDRG2 gene.Thus, the primers in the kit may comprise, consist essentially of, orconsist of primers for the purposes of amplifying methylated orunmethylated DNA (following bisulphite treatment). In one embodiment,the primers in the kit comprise, consist essentially of, or consist ofprimers which are capable of amplifying methylated and/or unmethylatedDNA following bisulfite treatment which DNA comprises, consistsessentially of, or consists of the nucleotide sequence set forth as SEQID NO: 524 and/or SEQ ID NO: 525.

The kit may alternatively or additionally employ bisulphite sequencingin order to determine the methylation status the NDRG2/NDRG4-family geneand in particular the NDRG4 and/or NDRG2 gene. Thus, the kit maycomprise primers for use in sequencing through the important CpG islandsin the NDRG2/NDRG4-family gene, in particular the NDRG4 and/or NDRG2gene. Thus, primers may be designed in both the sense and antisenseorientation to direct sequencing across the promoter region of the gene.In one embodiment, the primers in the kit comprise, consist essentiallyof, or consist of primers which are capable of sequencing of DNAfollowing bisulfite treatment which DNA comprises, consists essentiallyof, or consists of the nucleotide sequence set forth as SEQ ID NO: 524and/or SEQ ID NO: 525. Suitable primers are discussed herein in greaterdetail.

Similarly, the invention provides a kit for detecting a predispositionto, or the incidence of, a gastrointestinal cancer, such as colorectalcancer and/or gastric cancer and/or oesophageal cancer and in particularcolorectal cancer in a sample comprising:

(a) means for detecting an epigenetic change in at least one geneselected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, and MGMT, and/or atleast one gene selected from TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3and JAM3 (in all permutations and combinations including panels asdiscussed herein)(b) means for processing a faecal sample.

As discussed in more detail above, the at least one genemay be selectedfrom GATA4, OSMR, NDRG4 and SFRP2 since these genes provide aparticularly sensitive indication of colorectal cancer. The at least onegene may be selected from TFPI2, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3,in particular TFPI2.

The kit may comprise means for detecting an epigenetic change in a panelof genes comprising at least two, three, four, five or six of the genes,wherein detection of an epigenetic change in at least one of the genesin the panel is indicative of a predisposition to, or the incidence of,colorectal cancer or is used in one of the other application asdiscussed above. In one embodiment, the panel of genes comprises two,three, four, five or six genes.

In specific embodiments, the panel of genes comprises, consistsessentially of or consists of GATA4 and OSMR, GATA4 and NDRG4, GATA4 andSFRP2, OSMR and NDRG4, OSMR and SFRP2 or NDRG4 and SFRP2. In a morespecific embodiment, the panel of genes comprises, consists essentiallyof or consists of GATA4, OSMR and NDRG4, GATA4, OSMR and SFRP2, GATA4,NDRG4 and SFRP2 or OSMR, NDRG4 and SFRP2. Further panels comprise,consist essentially of or consist of GATA4, OSMR, NDRG4 and SFRP2.

An alternative panel of genes comprises, consists essentially of orconsists of NDRG4, OSMR, SFRP1, ADAM23, GATA5 and MGMT. The skilledperson would appreciate that other combinations and permutations may beformed as appropriate, as discussed in respect of the methods of theinvention.

In one embodiment, the means for processing a faecal sample comprise asealable vessel for collection of a faecal sample. Additionally oralternatively, the means for processing a faecal sample in the kitcomprises a homogenization buffer. The means for processing a faecalsample may further or alternatively comprise reagents forextraction/isolation/concentration/purification of DNA. Suitablereagents are known in the art and comprise, consist essentially of orconsist of alcohols such as ethanol and isopropanol for precipitation ofDNA. Salt-based precipitation may require high concentrations of saltsto precipitate contaminants. The salt may comprise, consist essentiallyof or consist of potassium acetate and/or ammonium acetate for example.Organic solvents may also be included in the kits to extractcontaminants from cell lysates. Thus, in one embodiment, the means forprocessing the faecal sample comprise, consist essentially of or consistof phenol, chloroform and isoamyl alcohol to extract the DNA. Suitablecombinations of reagents are envisaged as appropriate.

As discussed herein, which discussion applies mutatis mutandis,sensitivity of detection may be improved by increasing the quantity ofDNA in the sample. Accordingly, in one embodiment the means forprocessing a faecal sample comprises, consists essentially of orconsists of primers for directing amplification of DNA in the sample.Any suitable primers which amplify the at least one gene selected fromGATA4, OSMR, NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2,BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 may be utilised. Theprimers may not discriminate between methylated and unmethylated DNA(i.e. the primer binding sites lies outside of the CpG islands) thusproviding a general increase in the amount of DNA prior to determiningwhether the methylated form of the gene or genes is present in thesample.

Similarly, the invention provides a kit for detecting a predispositionto, or the incidence of, a gastrointestinal cancer, such as colorectalcancer and/or gastric cancer and/or oesophageal cancer and in particularcolorectal cancer in a sample comprising:

(a) means for detecting an epigenetic change in at least one geneselected from OSMR, SFRP1, NDRG4, GATA5, ADAM23, JPH3, SFRP2 and APCand/or at least one gene selected from TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3.

(b) means for processing a blood sample or derivative thereof.

As discussed in more detail above, the at least one gene may be selectedfrom OSMR, NDRG4 GATA5 and ADAM23 since these genes provide aparticularly sensitive indication of colorectal cancer in blood samples,or derivatives thereof and in particular plasma. The at least one genemay be selected from TFPI2, BNIP3, FOXE1, SYNE1, and SOX17, inparticular TFPI2.

The kit may comprise means for detecting an epigenetic change in a panelof genes comprising at least two, three, four, five or six of the genes,wherein detection of an epigenetic change in at least one of the genesin the panel is indicative of a predisposition to, or the incidence of,colorectal cancer or is used in one of the other application asdiscussed above. In one embodiment, the panel of genes comprises two,three, four, five or six genes.

In one embodiment, the panel of genes comprises, consists essentially ofor consists of OSMR, NDRG4, GATA5 and ADAM23. This kit may be used todiagnose early stage colorectal cancer, in particular stage 0 to IIcolorectal cancer.

In one embodiment, the means for processing a blood sample or derivativethereof comprises, consists essentially of or consists of a sealablevessel for collection of a blood sample. The means for processing ablood sample or derivative thereof may further or alternativelycomprises consists essentially of or consists of a reagents forextraction/isolation/concentration/purification of DNA. Suitablereagents are known in the art and comprise, consist essentially of orconsist of alcohols such as ethanol and isopropanol for precipitation ofDNA. Salt-based precipitation may require high concentrations of saltsto precipitate contaminants. The salt may comprise, consist essentiallyof or consist of potassium acetate and/or ammonium acetate for example.Organic solvents may also be included in the kits to extractcontaminants from cell lysates. Thus, in one embodiment, the means forprocessing the blood sample or derivative thereof comprise, consistessentially of or consist of phenol, chloroform and isoamyl alcohol toextract the DNA. Suitable combinations of reagents are envisaged asappropriate. The means for processing a blood sample or derivativethereof may comprise, consist essentially of or consist of isopropanol,magnetic beads or a silica-based membrane for isolating DNA. The meansfor processing a blood sample or derivative thereof may comprise,consist essentially of or consist of a kit as shown in table 1.

The means for processing a blood sample or derivative thereof, inparticular plasma or serum sample may comprise consist essentially of orconsist of one or more stabilizers. In one embodiment, stabilizers areincluded in the kit to be added to the blood sample, or derivativethereof. This is particularly relevant where the sample is not frozen.In one specific embodiment, where the sample is serum, stabilizers suchas stabilizers selected from EDTA and/or citrate and/or heparin areincluded. In a further embodiment, where the sample is plasma,stabilizers such as stabilizers selected from citrate and/or heparin maybe included. Antimicrobial agents such as antibiotics may be also beincluded in the kits of the invention prevent spoiling (of serum andplasma samples).

Similarly, the invention provides a kit for detecting a predispositionto, or the incidence of, a gastrointestinal cancer, such as colorectalcancer and/or gastric cancer and/or oesophageal cancer and in particularcolorectal cancer in a sample comprising:

(a) means for detecting an epigenetic change in at least one geneselected from GATA4, OSMR, NDRG4, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APCand MGMT

(b) means for processing a tissue sample, in particular a colon, rectalor appendix sample.

The kit may comprise means for detecting an epigenetic change in a panelof genes comprising at least two, three, four, five or six of the genes,wherein detection of an epigenetic change in at least one of the genesin the panel is indicative of a predisposition to, or the incidence of,a gastrointestinal cancer, such as colorectal cancer and/or gastriccancer and/or oesophageal cancer and in particular colorectal cancer oris used in one of the other applications as discussed above. In oneembodiment, the panel of genes comprises two, three, four, five or sixgenes.

In specific embodiments, the panel of genes comprises, consistsessentially of or consists of OSMR, GATA4 and ADAM23 or OSMR, GATA4 andGATA5.

These kits may also be useful in predicting the likelihood of successfultreatment of a gastrointestinal cancer, such as colorectal cancer and/orgastric cancer and/or oesophageal cancer and in particular colorectalcancer and/or the likelihood of resistance to treatment of agastrointestinal cancer, such as colorectal cancer and/or gastric cancerand/or oesophageal cancer and in particular colorectal cancer with a DNAdamaging agent and/or a DNA methyltransferase inhibitor and/or a HDACinhibitor, and/or selecting a suitable treatment regimen for agastrointestinal cancer, such as colorectal cancer and/or gastric cancerand/or oesophageal cancer and in particular colorectal cancer and/ordetermining the histopathological stage of a gastrointestinal cancer,such as colorectal cancer and/or gastric cancer and/or oesophagealcancer and in particular colorectal cancer in a sample, as discussed inrespect of the methods of the invention (which discussion appliesmutatis mutandis).

In a further embodiment, applicable to all relevant kits of theinvention, the means for detecting an epigenetic change in the panel ofgenes enable the detection to be carried out in a single reaction.Multiplexing is made possible for example through use of appropriatefluorophores having separable emission spectra. TaqMan probes, MolecularBeacons, Scorpions, etc . . . , as discussed herein, allow multiplemarkers to be measured in the same sample (multiplex PCR), sincefluorescent dyes with different emission spectra may be attached to thedifferent probes. Accordingly, suitably labelled probes and primers areencapsulated by the kits of the invention.

In a particularly preferred embodiment, the epigenetic change which isdetected using the kits of the invention is methylation. Many suitablereagents for methylation detection are known in the art, and arediscussed herein (which discussion applies here mutatis mutandis). Inparticular, hypermethylation of the promoter region of the gene(s) maybe detected using the kits of the invention. Thus, the means fordetecting methylation may comprise methylation specific PCR primers.Suitable primers may be selected from the primers comprising, consistingessentially of or consisting of the nucleotide sequences presented inany one of tables 2 to 18 as appropriate depending upon the kit and geneor genes concerned.

The kit may also include means for carrying out the methylation specificPCR in real time or at end point. The means for carrying out themethylation specific PCR/amplification in real time or at end point maycomprise hairpin primers (Amplifluor), hairpin probes (MolecularBeacons), hydrolytic probes (Taqman), FRET probe pairs (Lightcycler),primers incorporating a hairpin probe (Scorpion), fluorescent dyes (SYBRGreen etc.), DzyNA primers or oligonucleotide blockers for example.Suitable probes may be selected from the probes comprising, consistingessentially of or consisting of the nucleotide sequences presented intables 2 to 18 as appropriate for the respective genes. All appropriatecombinations are envisaged by the invention. Primers and probes fordetecting a suitable reference gene, such as beta-actin are displayed insome of these tables (3 and 4).

The end-point PCR fluorescence detection technique can use the sameapproaches as widely used for Real Time PCR-TaqMan assay, MolecularBeacons, Scorpion etc. Accordingly, the kits of the invention may, incertain embodiments, include means for carrying out end-pointmethylation specific PCR. The means for carrying out end-pointmethylation specific PCR/amplification may comprise primers and/orprobes as explained for PCR/amplification in Real-time.

In the real-time and end-point detection embodiments, the probes fordetection of amplification products may simply be used to monitorprogress of the amplification reaction in real-time and/or they may alsohave a role in determining the methylation status of the at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein), themselves. Thus,the probes may be designed in much the same fashion as the primers totake advantage of sequence differences following treatment with asuitable reagent such as sodium bisulphite dependent upon themethylation status of the appropriate cytosine residues (found in CpGdinucleotides).

The probes may comprise any suitable probe type for real-time detectionof amplification products as discussed above. Notably, however, with theAMPLIFLUOR and SCORPION embodiments, the probes are an integral part ofthe primers which are utilised. The probes are typically fluorescentlylabelled, although other label types may be utilised as appropriate(such as mass labels or radioisotope labels). These probes are alsosuitable for end-point detection.

The kits of the invention may be kits for use in MSP and in particularin a real-time or end point detection version of MSP.

The kits of the invention may incorporate reagents for quantification ofDNA such as those found in the Picogreen® dsDNA quantitation kitavailable from Molecular Probes, Invitrogen.

The kits of the invention may, additionally or alternatively comprise,consist essentially of or consist of a reagent which selectivelymodifies unmethylated cytosine residues in the DNA contained in thesample to produce detectable modified residues but which does not modifymethylated cytosine residues. The reagent preferably comprises, consistsessentially of or consists of a bisulphite reagent. The bisulphitereagent most preferably comprises, consists essentially of or consistsof sodium bisulphite. This reagent is capable of converting unmethylatedcytosine residues to uracil whereas methylated cytosines remainunconverted. This difference in residue may be utilised to distinguishbetween methylated and unmethylated nucleic acid in a downstreamprocess, such as PCR using primers which distinguish between cytosineand uracil (cytosine pairs with guanine, whereas uracil pairs withadenine). The reagent may be incorporated as the means for processing afaecal sample or means for processing a blood sample or derivativethereof depending upon the kit in question.

As discussed with respect to the methods of the invention, suitablecontrols may be utilised in order to act as quality control for themethods. Accordingly, in one embodiment, the kit of the inventionfurther comprises, consists essentially of or consists of one or morecontrol nucleic acid molecules of which the methylation status is known.These (one or more) control nucleic acid molecules may include bothnucleic acids which are known to be, or treated so as to be, methylatedand/or nucleic acid molecules which are known to be, or treated so as tobe, unmethylated. One example of a suitable internal reference gene,which is generally unmethylated, but may be treated so as to bemethylated, is β-actin.

Furthermore, the kit of the invention may further comprise, consistessentially of or consist of primers for the amplification of thecontrol nucleic acid. These primers may be the same primers as thoseutilised to monitor methylation in the test sample in specificembodiments. Thus, the control nucleic acid may comprise at least onegene selected from an NDRG2/NDRG4 subfamily gene (in particular NDRG4),GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3,FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in all permutations andcombinations including panels as discussed herein), for example takenfrom normal tissues in which it is known to be unmethylated. The controlnucleic acid may additionally comprise at least one gene selected froman NDRG2/NDRG4 subfamily gene (in particular NDRG4), GATA4, OSMR, GATA5,SFRP1, ADAM23, JPH3, SFRP2, APC, MGMT, TFPI2, BNIP3, FOXE1, SYNE1,SOX17, PHACTR3 and JAM3 (in all permutations and combinations includingpanels as discussed herein) in methylated form, for example asmethylated by a methyltransferase enzyme such as SssI methyltransferasefor example.

Suitable probes and/or oligonucleotide blockers for use in determiningthe methylation status of the control nucleic acid molecules may also beincorporated into the kits of the invention. The probes may comprise anysuitable probe type for real-time detection of amplification products.The discussion provided above applies mutatis mutandis.

The kits of the invention may additionally include suitable buffers andother reagents for carrying out the claimed methods of the invention.Thus, the discussion provided in respect of the methods of the inventionas to the requirements for determination of the methylation status of atleast one gene selected from an NDRG2/NDRG4 subfamily gene (inparticular NDRG4), GATA4, OSMR, GATA5, SFRP1, ADAM23, JPH3, SFRP2, APC,MGMT, TFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3 and JAM3 (in allpermutations and combinations including panels as discussed herein),apply mutatis mutandis here.

In specific embodiments, the kit of the invention further comprises,consists essentially of, or consists of nucleic acid amplificationbuffers. Suitable reagents may be selected from (NH₄)₂SO₄, Tris (pH8.8), MgCl₂, β-mercaptoethanol and stock solutions of dNTPs. Reagentsmay be supplied at any suitable concentration.

The kit may also additionally comprise, consist essentially of orconsist of enzymes to catalyze nucleic acid amplification. Thus, the kitmay also additionally comprise, consist essentially of or consist of asuitable polymerase for nucleic acid amplification. Examples includethose from both family A and family B type polymerases, such as Taq(such as the commercially available Jumpstart DNA Taq polymerase), Pfu,Vent etc.

The various components of the kit may be packaged separately in separatecompartments or may, for example be stored together where appropriate.

The kit may also incorporate suitable instructions for use, which may beprinted on a separate sheet or incorporated into the kit packaging forexample.

In one specific aspect, the methods and kits of the invention may becombined with the other methods and kits of the invention in order toprovide improved diagnosis, histopathological analysis, pharmacogenomicanalysis etc. of a gastrointestinal cancer, such as colorectal cancerand/or gastric cancer and/or oesophageal cancer and in particularcolorectal cancer.

Accordingly, all embodiments of the methods and kits of the inventionapply mutatis mutandis to the respective aspects of the invention.

The invention will now be described with respect to the followingnon-limiting examples.

DETAILED DESCRIPTION OF THE INVENTION Brief Description of the Drawings

FIG. 1a . NDRG4: Bisulfite sequencing of colorectal cancer tissue (T),normal colon mucosa (N), the methylated colorectal cancer cell line(HCT116) and the unmethylated cell line SW480. White and black squaresrepresent methylated and unmethylated CpG dinucleotides in NDRG4respectively. Each row represents a single clone. Location of the CpGare relative to the transcription start site. The location of the MSPprimers is positions 20 to 23 and 31 to 34 respectively.

FIG. 1b . NDRG2: Bisulfite sequencing of colon carcinoma cell lines (RKOand LS174T). White and black squares represent methylated andunmethylated CpG dinucleotides in NDRG2B respectively.

FIG. 2 shows relative expression of NDRG4 after treatment with DAC andTSA compared to untreated cell lines. Cyclophilin was used as areference gene for expression normalisation.

FIG. 3a shows methylated NDRG4 sequence (SEQ ID NO: 524)(NM_020465:-1000to +1000 relative to TSS) Bisulfite sequence primers in mid-grey; Flankprimers for the nested MSP are underlined; Methylated MSP primers inlight-grey; Unmethylated primers in dark-grey and light-grey

FIG. 3b shows methylated NDRG2 sequence (SEQ ID NO: 525). Bisulfitesequence primers in mid-grey; Flank primers for the nested MSP areunderlined; Methylated MSP primers in light-grey; Unmethylated primersin dark-grey and light-grey

FIG. 4 shows sentivity of different markers, with 100% specificity. Xaxis=: % positive in real time QMSP; Y axis=: different markers. Case:n=65 carcinoma's; Controls: n=33 histologically normal resection ends.

FIG. 5 shows a decision tree for determination of the methylation statusof the gene of interest linked to colorectal cancer in clinical samples(real-time MSP).

FIG. 6 presents results of real-time MSP carried out on 9 differentgenes for 34 colon carcinoma tissue samples, 16 colon adenoma tissuesamples and 63 breast (20), lung (21) and bladder (22) cancer samples.Sensitivity performance for each gene is shown wherein the analyticalcut-off was set to give 100% specificity (based on the non-cancerouscontrols).

FIG. 7 presents results of real-time MSP carried out on 10 differentgenes for 34 colon carcinoma tissue samples, 16 colon adenoma tissuesamples and 59 samples from patients with cancer other than CRC.Sensitivity performance for each gene is shown wherein the analyticalcut-off was set to give 100% specificity (based on the non-cancerouscontrols), except for JPH3 where 95% specificity was obtained.

FIG. 8 presents results of real-time MSP carried out on 8 differentgenes for plasma training set 1 and 5 different genes for plasmatraining set 2. Plasma training set 1 includes 34 samples with nosuspicious findings, 25 samples from patients with cancers other thancolon and 42 samples from patients covering all stages of CRC, with 81%representing stages I-III of disease. Plasma training set 2 was testedon 64 samples with no suspicious findings, 49 adenomas, 25 samples frompatients with cancer other than colon cancer and 78 samples frompatients covering all stages of CRC, with 76% representing stages I-IIIof disease.

FIG. 9 is an overview of the NDRG4 study showing the patient groupswhich were investigated.

FIG. 10 shows a schematic representation of the promoter region ofNDRG4. A dense CpG island from −556 to +869 relative to thetranscription start site (TSS) (indicated by a curved arrow) is shown.Locations of CpG dinucleotides (representated by 1), ORF NDRG4 (asindicated with a grey rectangle) and the region of the hypermethylatedfragment identified by Methylation Specific PCR (MSP), Quantative MSP(qMSP) and Bisulfite sequencing (BS) primers are indicated.

FIG. 11a shows results of methylation specific PCR (MSP) with primerpair 2 to detect DNA methylation in eight different CRC cell lines.

FIG. 11b shows bisulfite sequencing of two CRC cell lines, namely HCT116and SW480. Six different clones were sequenced. Each row represents anindividual cloned allele that was sequenced following sodium bisulfiteDNA modification. Each box indicate a CpG dinucleotide (black box;methylated CpG site, white box; unmethylated CpG site)

FIG. 11c shows NDRG4 expression in colon cancer cell lines (RKO andHCT116) after treatment with the methylation inhibitor5-aza-2′-doxycytidine (DAC).

FIG. 12a shows bisulfite sequencing of three cases of cancers (T) andtheir matched normal non malignant mucosa tissue (N). Six differentclones were sequenced.

FIG. 12b shows levels of NDRG4 transcript expression measured byrealtime PCR in colon cancer tissue (labelled for T) and matched normalcolon tissue samples (labelled for N) for three different persons. Foreach patient, levels of NDRG4 expression in the normal mucosa tissuewere set to equal 1. The experiments were performed three times.

FIG. 12c shows localization of NDRG4 expression. Immunohistochemicalstaining of NDRG4 in normal mucosa and colon tumor shows no staining incancer cells but clear staining in the nuclei of normal epithelialcells.

EXPERIMENTAL SECTION

1) NDRG Experiments

Cell Culture

Colon cancer cell lines LS174T, HCT116, HT29, RKO, CaCo2, Colo205, SW48and SW480 were used for MSP, bisulfite sequencing and real time(reexpression) RT-PCR (1 MM DAC and 300 nM TSA).

Study Population

Formalin-fixed, paraffin-embedded colon mucosa tissue of colorectalcancer patients and controls over 50 years of age was retrospectivelycollected from the archive of the dept. of Pathology of the UniversityHospital Maastricht. Approval was obtained by the Medical EthicalCommittee (MEC) of the Maastricht University and the University HospitalMaastricht. If present, also normal and adenoma tissue was collectedfrom these cases. The control group consists of histologically normalbiopsy material from patients undergoing endoscopy because ofnon-specific abdominal complaints, adenoma biopsies from patients whichdid not develop colorectal cancers within 5-10 years. Colorectal cancerspatients and controls were excluded if being diagnosed with additionalcancers other than non-melanoma skin cancer.

Methylation-Specific PCR

DNA methylation in the CpG islands of the gene promoter was determinedby bisulfite treatment of genomic DNA with sodium bisulfite followed byMSP. Briefly, bisulfite modification of genomic DNA was carried usingthe EZ DNA methylation kit (Zymo Research). MSP analysis on DNAretrieved from formalin-fixed, paraffin embedded tissue was facilitatedby first amplifying the DNA with flanking PCR primers which amplifybisulfite-modified DNA but do not make the distinction betweenmethylated or unmethylated DNA. This PCR product was used as a templatefor the MSP reaction. All PCRs were performed with controls forunmethylated DNA (DNA from normal lymfocytes), methylated DNA (normallymphocyte DNA treated in vitro with SssI methyltransferase (New EnglandBiolabs)), and a control without DNA. Ten μl of each MSP reaction weredirectly loaded onto 2% agarose visualized under UV illumination. Primersequences and PCR conditions, are specified in Table 19.

Alternatively, DNA methylation was determined by QMSP.

TABLE 19 NDRG4 and NDRG2b MSP primers SEQ ID Primer Ann. No Gene nameSequence 5′-3 Size Temp. Cycles Posn.*  5 NDRG4 Flank Fggttygttygggattagttttagg 155 56 35 -144  6 NDRG4 Flank Rcraacaaccaaaaacccctc bp  +10  7 NDRG4 U sensegattagttttaggtttggtattgttttgt 100 66 25 -133 bp  8 NDRG4 Uaaaaccaaactaaaaacaatacacca  -34 antisense  9 NDRG4 M sensetttaggttcggtatcgtttcgc  88 66 25 -126 10 NDRG4 M cgaactaaaaacgatacgccgbp  -39 antisense 20 NDRG2 Flank F YGTTTTTTATTTATAGYGGTTTTT 21 NDRG2Flank R TCCTAATACCTCTCCTCTCTTTACTAC 22 NDRG2 U senseTTTTATTTATAGTGGTTTTTTGTATTTTTT 23 NDRG2 U TCTCCTCTCTTTACTACATCCCAACAantisense 24 NDRG2 M sense TTTATAGCGGTTTTTCGTATTTTTC 25 NDRG2 MCCTCTCTTTACTACGTCCCGACG antisense *Position relative to transcriptionstart siteBisulfite Genomic Sequencing

Genomic DNA was isolated using the Wizard Genomic DNA Purification kit(Promega, Leiden, the Netherlands). Bisulfite modification of genomicDNA was carried out using the EZ DNA methylation kit (Zymo Research).PCR products were subcloned using the TA cloning kit (Invitrogen, Breda,the Netherlands) and single colonies were selected and sequenced. Primersequences and PCR conditions are specified in Table 20.

TABLE 20 NDRG4 and NDRG2b bisulfite sequencing primers SEQ ID PrimerAnn. NO Gene name Sequence 5′-3 Size Temp. Cycles Position 569 NDRG4 Fgatyggggtgttttttaggttt 262 64 40 -251 bp   6 NDRG4 Rcraacaaccaaaaacccctc  +10 522 NDRG2 F TTTGTTGGTTATTTTTTTTTTATTTTT 523NDRG2 R CCCCCAAACTCAATAATAAAAACReal-Time RT-PCR

Total RNA isolation was isolated by use of the Rneasy Mini kit (Qiagen)cDNA synthesis using the Iscript cDNA synthesis kit (Bio-Rad).Quantitative real-time reverse transcription-PCR was done using SYBRGreen PCR Master Mix (Applied Biosystems, Nieuwekerk a/d IJssel, theNetherlands). Primers and PCR conditions are specified in Table 21.

TABLE 21 NDRG4 Real time RT-PCR primers SEQ ID Primer Ann. NO Gene nameSequence 5′-3 Size Temp. Cycles 1 NDRG4 F cctgaggagaagccgctg 101 bp 6040 2 NDRG4 R atgtcatgttccttccagtctgtExpression Analysis of NDRG4

Expression of the NDRG4 gene was determined by real-timereverse-transcription PCR (RT-PCR). The NDRG4 gene was found to be wellexpressed in normal colon cell lines, whereas it was not expressed inthe colon cancer cell lines. Since this on its own did not indicate thatthe silencing is epigenetic, the RKO and HCT116 cell lines were treatedwith the reagent DAC (5′dazacytidine) and TSA. Relative expression ofNDRG4 after treatment with DAC and TSA was compared to untreated celllines. Cyclophilin was used as a reference gene for expressionnormalisation. FIG. 2 shows that treatment resulted in a reactivation ofNDRG4 expression, providing evidence for epigenetic silencing of thegene in colon cancer cells.

CpG Island Methylation Status Analysis of NDRG4 and NDRG2

Having observed that the silencing of NDRG4 expression was reversedafter treatment with DAC and TSA, the association between thetranscriptional inactivation and the putative epigenetic aberration wasfurther investigated. The NDRG CpG island methylation status wasestablished by PCR analysis of bisulfite-modified genomic DNA, whichinduces chemical conversion of unmethylated, but not methylated,cytosine to uracil, using the procedures as specified.

Table V shows that NDRG4 CpG island methylation analysed by MSP wasobserved in the cancer cell lines LS174T, HCT116, HT29, RKO, CaCO2 andSW48, whereas it was absent in the unmethylated cell line SW480.

Similarly, NDRG2 CpG island methylation analysed by MSP with differentprimer sets (a to d) was observed in most of the cancer cell lines. Inall cancer cell lines LS174T, HCT116, HT29, RKO, CaCO2 and SW48, NDRG2CpG island methylation was observed with primer sets b of table 19.

TABLE 22 Methylation status of colorectal cancer cell lines (analysed byMSP) Colo20 LS174T HCT116 HT29 RKO CaC02 5 SW48 SW480 NDRG4 M M M M M MM U NDRG2 a U U U U U U U / NDRG2b U M U M M M M / NDRG2c U M U M M M MU? NDRG2d (a) U M U M M U U M

Following the demonstration of the epigenetic loss of function of NDRG4in cancer-cell lines, we assessed the prevalence of NDRG4 CpG islandpromoter hypermethylation in cancer patients. As expected, NDRG4 CpGisland promoter hypermethylation was absent in normal mucosa frompatients without cancer. As indicated in Table 23, NDRG4 CpG islandpromoter hypermethylation was observed with different frequency amongeach class of neoplasm. NDRG4 was methylated in 76% of the 88investigated carcinoma tissues and in 57% of 57 adenomas with concurrentcolorectal cancer. In adenomas from patients that did not havecolorectal cancer (low-grade dysplastic non-progressed adenomas), NDRG4methylation was significantly lower (14%), indicating the prognosticvalue of this NDRG4 methylation towards colorectal cancer development

TABLE 23 Prevalence of NDRG4 methylation in colorectal tissueMethylation (%) Morphologically normal mucosa adjacent to tumor 2.5 (n =82) tissue Adenomas from patients also presenting a colorectal 57 (n =57) carcinoma Carcinoma tissue 76 (n = 88) Normal mucosa from patientswithout cancer  0 (n = 27) Adenomas from patients that did not develop14 (n = 51) colorectal cancer (low-grade dysplastic non- progressedadenomas)NDRG4 Methylation Compared to Methylation of Other Markers

Samples from resected tumors and histologically normal resection weretested for hypermethylation of 13 genes. Representative results areshown in FIG. 4. The highest methylation was obtained for SFRP1, SFRP2,NDRG4, GATA4 and GATA5. All showed a sensitivity >40% for 100%specificity. We tested the ability of the NDRG4 methylation marker toimprove the sensitivity of cancer detection with a number of methylationmarkers selected on their ability to detect colorectal cancer. The othergenes were selected from the group consisting of SFRP1, SFRP2, GATA-4,GATA-5, CHFR, APC(2), MGMT, p16, Vimentin, p14, RASSF1a and RAB32. In afirst instance, the ability of NDRG4 to complement SFRP1 was analysed.30% of colon carcinoma samples (n=18) for which SFRP1 failed to behypermethylated, showed hypermethylation for NDRG4 (n=6). Similarly,carcinoma samples which failed to be detected by way of SFRP2, GATA4, orGATA5 methylation analysis, showed hypermethylation for NDRG4. In fact,the combination of NDRG4 with any of the methylation markers from FIG. 4improved diagnosis of cancer

NDRG-4 MSP on Other Cancer Types (Methylated Cancers)

NDRG-4 methylation was assessed on other cancer types showinghypermethylation for certain genes. These cancer types comprisedmelanoma, clear cell kidney cancer, ovarian carcinoma, prostate cancer,breast cancer and gastric cancer. The results were as follows:

Melanoma: 0 out of 8 samples were methylated for NDRG4

Clear cell kidney cancer: only 1 out of 10 samples was methylated forNDRG4

Ovarium carcinoma: 0 out of 20 samples were methylated for NDRG4

Prostate cancer: 0 out of 10 samples were methylated for NDRG4

Breast cancer: 0/7 lobular cancers and 0/9 Ductal cancers weremethylated for NDRG4

In contrast to these results, in all of the 6 gastric cancers testedmethylation for NDRG4 was observed. This seems to indicate that NDRG4 isa type-specific cancer methylation marker and is preferably used todetect colon cancer and/or gastric cancer.

REFERENCES

-   Akey, D. T., Akey, J. M., Zhang, K., Jin, L., 2002. Genomics,    80:376-384.-   Angela Di Vinci, Ilaria Gelvi, Barbara Banelli, Ida Casciano,    Giorgio Allemanni and Massimo Romani. Laboratory    Investigation (2006) 1-7-   Barringer K J, Orgel L, Wahl G, Gingeras T R. Gene. 1990 Apr. 30;    89(1):117-22-   Boggs B. A., Cheung P, Heard E, Spector D L, Chinault A C, Allis    C D. Nat. Genet. 2002, 30: 73-76.-   Compton, J. Nature. 1991 Mar. 7; 350(6313):91-2.-   Cottrell, S., Distler, J., Goodman, N., Mooney, S., Kluth, A., Olek,    A., Schwope, I., Tetzner, R., Ziebarth, H., Berlin, K. Nucleic Acid    Res. 2004, 32:E10.-   Cross, S H et al. Nature Genetics 1994, 6, 236-244;-   Deng Y, Yao L, Chau L, Ng S Svb, Peng Y, Liu X, Au W S, Wang J, Li    F, Ji S, Han H, Nie X, Li Q, Kung H F, Leung S Y, Lin M C. Int J    Cancer. 2003, 106(6):984.-   Eads, C. A., Danenberg, K. D., Kawakami, K, Saltz, L. B., Blake C.,    shibata, D; Danenberg, P. V. and Laird P. W. Nucleic acid Res. 2000,    28: E32-   Fahy E, Kwoh D Y, Gingeras T R. PCR Methods Appl. 1991 August;    1(1):25-33-   Furuichi Y, Wataya Y, Hayatsu H, Ukita T. Biochem Biophys Res    Commun. 1970 Dec. 9; 41(5):1185-91-   Guan R J, Ford H L, Fu Y, Li Y, Shaw L M, Pardee A B. Cancer Res.    2000, 60(3):749-55.-   Herman J G, Graff J R, Myohanen S, Nelkin B D, Baylin S B. Proc.    Natl. Acad. Sci. USA. 1996: 93(18):9821-9826-   Hu X L, Liu X P, Lin S X, Deng Y C, Liu N, Li X, Yao L B. World J    Gastroenterol. 2004, 10(23):3518-21-   Johnstone R. W. Nat. Rev. Drug Discov. 2002, 1: 287-299.-   Jones P A and Baylin S B Nat. Rev. Genet. 2002, 3: 415-428.-   Jorgensen, H F., Adie, K., Chaubert, P. and Bird A. Nucleic Acids    Research, 2006, Vol. 34, No. 13 e96-   Kondo Y, shen L, Issa J P Mol. Cell. Biol. 2003, 23: 206-215.-   Lund A H, and van Lohuizen M. Genes Dev. 2004, 18: 2315-2335.-   Lusis E A, Watson M A, Chicoine M R, Lyman M, Roerig P, Reifenberger    G, Gutmann D H, Perry A. Cancer Res. 2005, 65(16):7121-6.-   Mitchelmore C, Buchmann-Moller S, Rask L, West M J, Troncoso J C,    Jensen N A. Neurobiol Dis. 2004, 16(1):48-58-   Nishimoto S, Tawara J, Toyoda H, Kitamura K, Komurasaki T. Eur J    Biochem. 2003 June; 270(11):2521-31-   Qu X, Zhai Y, Wei H, Zhang C, Xing G, Yu Y, He F. Mol Cell Biochem.    2002, 229(1-2):35-44.-   Rand K., Qu, W., Ho, T., Clark, S. J., Molloy, P. Methods. 2002,    27:114-120.-   Sasaki, M., Anast, J., Bassett, W., Kawakami, T., Sakuragi, N., and    Dahiya, R. Biochem. Biophys. Res. Commun. 2003, 209: 305-309.-   Shiio Y, Eisenman R N Proc. Natl. Acad. Sci. USA. 2003, 100:    7357-7362.-   Shiraisi, M et al. Biol Chem. 1999, 380(9):1127-1131-   Zhao W, Tang R, Huang Y, Wang W, Zhou Z, Gu S, Dai J, Ying K, Xie Y,    Mao Y. Biochim Biophys Acta. 2001, 1519(1-2):134-138;    2) Experiments on Faecal DNA

Example 1

Materials and Methods in Relation to Faecal DNA

Sample Collection and Processing

A standardized multicenter screening trial (The Netherlands) wasinitiated in 2006. In this trial, non symptomatic subjects aged 50 orabove are screened with colonoscopy, FOBT and real-time MSP using DNAfrom stool and blood. In addition, prospectively collected stool samplesfrom multiple centers (Germany and The Netherlands) were used. In thesetrials, symptomatic patients, attending a Gastroenterology clinic andultimately diagnosed with CRC, provided a stool sample for use inreal-time MSP. From the ongoing trials 147 stool samples were availablefor the present study. 3 main categories of stool samples were used: 67samples with no suspicious findings, 58 adenomas and 22 samples frompatients covering all stages of CRC, with 90% representing early stagedisease.

After defecation in a special bucket, patients added 250 ml of stoolhomogenization buffer (Amresco, Solon, Ohio, USA) to the sample. Sampleswere shipped to the laboratory and further processed within 72 hoursafter defecation. Stool homogenization buffer was added to a ratio 1:7,and the samples were homogenized and aliquoted in portions of 32 ml.

DNA Extraction from Stool

Single aliquots (32 ml containing the equivalent of 4 g of stool) werecentrifuged for 5 minutes at 2540 rcf at 20° C. The supernatant wasretained and centrifuged a second time (10 minutes at 16500 rcf at 4°C.). 22 ml of the supernatant obtained following the secondcentrifugation step was incubated with 5 μl Rnase A for 60 minutes at37° C. Total DNA was then SodiumAcetate (pH 5.2)—isopropanolprecipitated and washed with 70% ethanol. The DNA was resuspended in 4ml 1×TE (pH 7.4). 400 μl 10× buffer (240 mM EDTA (pH=8.0), 750 mM NaC),400 μl 10% SDS, and 20 μl Proteinase K (20 mg/ml) was added and thesamples were incubated at 48° C. overnight at constant shaking (225RPM). After centrifugation (3000 RCF for 30 seconds at roomtemperature), 5 ml of Phenol: Chloroform: Isoamylalcohol (25:24:1, v/v;Invitrogen) was added and incubated for 10 minutes at room temperatureshaking at 225 RPM and centrifuged for 5 minutes at 3000 RCF. Theaqueous layer was transferred to a new tube containing 5 ml of Phenol:Chloroform: Isoamylalcohol. Again, the samples were incubated for 10minutes at room temperature shaking at 225 RPM and centrifuged for 5minutes at 3000 RCF at room temperature. The aqueous layer wastransferred to a new tube and DNA was precipitated by adding 500 μl 7.5M Ammonium Acetate, 5 μl glycogen and 10 ml of cold 100% Ethanol (−20°C.), further incubated at −20° C. for at least 1 hour and centrifuged at15000 RCF for 30 minutes at 4° C. Pellets were washed with 3.5 mlfreshly prepared 70% Ethanol and air dried. Pellets were finalyresuspended in 2 ml of LoTE pH 8.0 and stored at −80° C., until furtherprocessing. Average yield of DNA was 462 μg (ranging from 46-2127 μg; SD420)

DNA Modification

An upscaled DNA modification step was applied to 32 μg of the obtainedDNA. 16 Aliquots of 2 μg of DNA were subjected to bisulfite modificationin 96-wells format on a pipetting robot (Tecan), using the EZ-96DNAMethylation kit (Zymo Research), according to the manufacturer'sprotocol. Basically, aliquots of 45 μl were mixed with 5 μl ofM-Dilution Buffer and incubated at 37° C. for 15 minutes shaking at 1100rpm. Then 100 μl of the diluted CT Conversion Reagent was added andsamples were incubated at 70° C. for 3 hours, shaking at 1100 rpm in thedark. After conversion, the samples were desalted by incubation on icefor 10 minutes and addition of 400 μl of M-Binding buffer. The sampleswere loaded on a Zymo-Spin I Column in a collection tube and aftercentrifugation washed with 200 μl of M-Wash Buffer. 200 μl ofM-Desulphonation Buffer was put onto the column and incubated at roomtemperature for 15 minutes. After centrifugation of the columns, theywere washed twice with 200 μl of M-Wash Buffer. Finally, the DNA waswashed from the column in 50 μl Tris-HCl 1 mM pH8.0 and stored at −80°C., until further processing.

DNA Concentration

Bisulfite treated DNA is concentrated using the ZYMO Clean andConcentrator Kit (Zymo Research). To each aliquot of DNA 100 μl of DNABinding Buffer was added. The equivalent of ˜6 μg of DNA (quantifiedbefore bisulfite treatment) was transferred to a Zymo-Spin™ Column in acollection tube. (16 wells with bisulfite treated DNA per sample aredivided over 5 Zymo-Spin™ columns.) The tubes were centrifugedat >10,000 rpm for 30 seconds and washed twice with 200 μl of washbuffer. The DNA was eluted of the column by adding 6 μl of 1 mMTris-HCl, pH=8.0, incubated for 1 minute and centrifugation at >10,000rpm for 30 seconds. The eluates of columns with the same sample werepooled. The resulting chemical treated DNA was used as template forreal-time MSP.

DNA Amplification

Real-time MSP was applied on a 7900HT fast real-time PCR system (AppliedBiosystems). 2.4 μl of the modified DNA (equivalent to 2.5 μgunconverted DNA) was added to a PCR mix (total volume 12 μl) containingbuffer (16.6 mM (NH4)2SO4, 67 mM Tris (pH 8.8), 6.7 mM MgCl2, 10 mMβ-mercaptoethanol), dNTPs (5 mM), forward primer (6 ng), reverse primer(18 ng), molecular beacon (0.16 μM), BSA (0.1 μg), and Jumpstart DNA Taqpolymerase (0.4 units; Sigma Aldrich). The primer sequences andmolecular beacon sequences used for each of the genes are summarized intable 1. Cycle program used was as follows: 5 minutes 95° C., followedby 45 cycles of 30 seconds 95° C., 30 seconds 57° C. (51° C. for APC),and 30 seconds 72° C., followed by 5 minutes 72° C. A standard curve(2×106−20 copies) was included to determine copy numbers of unknownsamples by interpolation of their Ct values to the standard curve.

Results

Marker Identification and Validation in Colon Tissue Samples.

Assay Validity Rate in Tissue and Stool:

230 FFPE and 147 stool samples were processed using real-time MSP. Thereal-time MSP assays produced valid results in 99% of the FFPE and stoolsamples.

Marker Selection in Colon Tissue:

Based on re-expression, 224 different gene assays representing 145 genepromotors were tested on the Base5 methylation profiling platform (datanot shown, see reference 1 for details). The 37 most differentiallymethylated gene sequences assessing 29 gene promoters were validated onretrospectively collected tumors from 65 colorectal cancer patients (allstages) and 74 distant resection ends (histopathologically normal) usingreal-time MSP. Several markers reliably detected CRC in those tissuesamples (data not shown). The results were confirmed on an independenttest set containing 39 tissue controls (non-cancerous), 34 carcinomasand 16 adenomas. Several combinations of the tested markers reliablydetected CRC with high specificity and sensitivity.

The ten best performing markers GATA5, GATA4, SFRP1, SFRP2, APC, MGMT,NDRG4, OSMR, JPH3 and ADAM23 were validated with primer sets and beaconprobes as specified in Table 24. In addition to the colon test genes,the independent reference gene β-Actin (ACT) was also measured. Theratios between the colon test genes and ACT were calculated, and are thetest result of the assay. The samples were classified as methylated,non-methylated, or invalid based on the decision tree shown in FIG. 5.

The individual performance of the ten markers is shown in Table 25.Dependent on the cutoff applied, different sensitivities were obtainedfor the individual markers. For 100% specificity of the marker,sensitivities (%) ranged from 56 to 66 for GATA5, 78 to 82 GATA4, 84 to92 for SFRP1, 72 to 84 for SFRP2, 40 to 46 for APC, 44 for MGMT, 64 to66 for NDRG4, 88 for OSMR, 82 for JPH3 and 50 for ADAM23.

TABLE 24 Primers sequences and beacon sequences SEQ ID NO: 26 β-Actinforward primer 5′- TAGGGAGTATATAGGTTGGGGAAGTT-3′ 27 reverse primer 5′-AACACACAATAACAAACACAAATTCAC-3′ 28 beacon 5′-FAM-CGACTGCGTGTGGGGTGGTGATGGAGG AGGTTTAGGCAGTCG-3′-DABCYL 29 GATA4forward primer 5′-AGGTTAGTTAGCGTTTTAGGGTC-3′ 30 reverse primer5′-ACGACGACGAAACCTCTCG-3′ 31 beacon 5′-FAM- CGACATGCCTCGCGACTCGAATCCCCGACCCAGCATGTCG-3′-DABCYL 32 GATA5 forward primer5′-AGTTCGTTTTTAGGTTAGTTTTCGGC-3′ 33 reverse primer5′-CCAATACAACTAAACGAACGAACCG-3′ 34 beacon 5′-FAM-CGACATGCGTAGGGAGGTAGAGGGGTT CGGGATTCCGTAGCATGTCG-3′-DABCYL 35 SFRP1forward primer 5′-TGTAGTTTTCGGAGTTAGTGTCGCGC-3′ 36 reverse primer5′-CCTACGATCGAAAACGACGCGAACG-3′ beacon 5′-FAM-CGACATGCTCGGGAGTCGGGGCGTATT TAGTTCGTAGCGGCATGTCG-3′-DABCYL 38 SFR2forward primer 5′-GGGTCGGAGTTTTTCGGAGTTGCGC-3′ 39 reverse primer5′-CCGCTCTCTTCGCTAAATACGACTCG-3′ 40 beacon 5′-FAM-CGACATGCGGTGTTTCGTTTTTTCGCGT TTTAGTCGTCGGGCATGTCG-3′-DABCYL 17 NDRG4forward primer 5′-GTATTTTAGTCGCGTAGAAGGC-3′ 18 reverse primer5′-AATTTAACGAATATAAACGCTCGAC-3′ 19 beacon 5′-FAM-CGACATGCCCGAACGAACCGCGATCCC TGCATGTCG-3′-DABCYL 41 APC forward primer5′-GAACCAAAACGCTCCCCAT-3′ 42 reverse primer5′-TTATATGTCGGTTACGTGCGTTTATAT-3′ 43 beacon 5′-FAM-CGTCTGCCCCGTCGAAAACCCGCCGAT TAACGCAGACG-3′-DABCYL 44 ADAM23forward primer 5′-GAAGGACGAGAAGTAGGCG-3′ 45 reverse primer5′-CTAACGAACTACAACCTTACCGA-3′ 46 beacon 5′-FAM-CGACATGCCCCCGACCCGCACGCCGCC CTGCATGTCG-3′-DABCYL 47 OSMR(3)forward primer 5′-TTTGGTCGGGGTAGGAGTAGC-3′ 48 reverse primer5′-CGAACTTTACGAACAACGAAC-3′ 49 beacon 5′-FAM-CGACATGCCCGTACCCCGCGCGCAGCA TGTCG-3′-DABCYL 47 OSMR(4) forward primer5′-TTTGGTCGGGGTAGGAGTAGC-3′ 50 reverse primer5′-AAAAACTTAAAAACCGAAAACTCG-3′ 49 beacon 5′-FAM-CGACATGCCCGTACCCCGCGCGCAGCA TGTCG-3′-DABCYL 51 JPH3 forward primer5′-TTAGATTTCGTAAACGGTGAAAAC-3′ 52 reverse primer5′-TCTCCTCCGAAAAACGCTC-3′ 53 beacon 5′-FAM- CGTCTGCAACCGCCGACGACCGCGACGCAGACG-3′-DABCYL 54 MGMT forward primer 5′-TTTCGACGTTCGTAGGTTTTCGC-3′ 55reverse primer 5′-GCACTCTTCCGAAAACGAAACG-3′ 56 beacon 5′-FAM-CGTCTCGCGTGCGTATCGTTTGCGATTT GGTGAGTGTTTGGGGCGAGACG-3′-DABCYL

TABLE 25 Individual performance of markers on adenoma and carcinomacolorectal tissue samples Cases (adenoma + Cut off SensitivitySpecificity Gene* carcinoma) Controls ratio** (%) (%) GATA5 50 39 12 (5)56 (66) 100 GATA4 50 39 17 (12) 78 (82) 100 SFRP1 50 39 47 (25) 84 (92)100 SFRP2 50 39 28 (9) 72 (84) 100 APC 50 39 16 (5) 40 (46) 100 MGMT 5039 18 44 100 NDRG4 50 39  7 (1) 64 (66) 100 OSMR 50 39 47 88 100 (3)JPH3 50 39 55 (75) 82 (82) 95 (100) ADAM23 50 39  2 50 100 *(3) reflectsthe primer combinations used for assessing methylation of the OSMR gene**In case two sets of cut off ratio were assessed, the second set andits corresponding sensitivity is indicated between ( ).Complementarity of Markers

The different markers were tested on their complementarity. Severalcombinations of the tested markers reliably detected CRC with highspecificity and sensitivity. Results are summarized in Table 26. For100% specificity, sensitivities (%) ranged between 90 to 98 forcombinations of two markers. A sensitivity of 100% was obtained for the3-marker combinations SFRP1+SFRP2+APC and SFRP2+OSMR+APC.

TABLE 26 Complementarity of markers on adenoma and carcinoma colorectaltissue samples Genes* Sensitivity** Specificity NDRG4 + OSMR (4) 90%100% SFRP2 + APC 92% 100% APC + OSMR (3) 92% 100% MGMT + OSMR (3) 92%100% OSMR (3) + OSMR (4) 92% 100% SFRP1 + APC 94% 100% SFRP1 + GATA-494% 100% SFRP1 + NDRG4 94% 100% SFRP1 + OSMR (3) 94% 100% SFRP1 + OSMR(4) 94% 100% GATA-4 + OSMR (4) 94% 100% NDRG4 + OSMR (3) 94% 100%GATA-5 + SFRP1 96% 100% GATA-5 + OSMR (3) 96% 100% SFRP2 + OSMR (4) 96%100% GATA-4 + OSMR (3) 96% 100% SFRP1 + SFRP2 98% 100% SFRP2 + OSMR (3)98% 100% SFRP1 + SFRP2 + APC 100% 100% SFRP2 + OSMR (3) + APC 100% 100%*(3) and (4) reflect the primer combinations used for assessingmethylation of the OSMR gene **Sensitivity corresponding to the secondcutoff set specified between ( ) in Table 25.Performance of Markers on Adenoma and Carcinoma Tissue Samples

Important for early cancer detection is the performance of the markerson early stage cancers. Therefore, the 50 cancer cases from the test setwere further divided into 2 diagnosis groups: carcinomas and adenomas.Results are summarized in table 27 and 28. Sensitivity for carcinomasranged from 35% to 88% for detection of colorectal cancer whereassensitivity for adenomas ranged from 31% to 88% both with acorresponding specificity of 100%. These results indicate that theselected set of genes are highly specific for colorectal cancer andinclude some promising early stage detection markers.

TABLE 27 Performance of the markers on carcinoma samples Cut off Gene*Carcinoma Controls ratio Sensitivity Specificity GATA5 34 39 12 53 100GATA4 34 39 17 74 100 SFRP1 34 39 47 82 100 SFRP2 34 39 28 68 100 APC 3439 16 35 100 MGMT 34 39 18 35 100 NDRG4 34 39 7 62 100 OSMR (3) 34 39 4788 100 JPH3 34 39 55 82 100 ADAM23 34 39 2 59 100 *(3) reflects theprimer combinations used for assessing methylation of the OSMR gene

TABLE 28 Performance of the markers on adenoma samples Cut off Gene*adenoma Controls ratio Sensitivity Specificity GATA5 16 39 12 63 100GATA4 16 39 17 88 100 SFRP1 16 39 47 88 100 SFRP2 16 39 28 81 100 APC 1639 16 50 100 MGMT 16 39 18 63 100 NDRG4 16 39 7 69 100 OSMR (3) 16 39 4788 100 JPH3 16 39 55 81 100 ADAM23 16 39 2 31 100 *(3) reflects theprimer combinations used for assessing methylation of the OSMR genePerformance of Markers in Fecal Samples

Nine of the best performing methylation markers in tissue (GATA4, GATA5,SFRP1, SFRP2, NDRG4, APC, ADAM23, OSMR3, and JPH3) were chosen to beevaluated in fecal samples. β-Actin copy numbers were also quantified asa control for sample quality and DNA yield. Methylated copies of thesegenes were quantified in all available stool samples by real-time MSP ona 7900HT fast real-time PCR system (Applied Biosystems).

The individual performance of the 9 genes (Actin, SFRP2, GATA5, GATA4,APC, SFRP1, NDRG4, OSMR3 and ADAM23) in fecal samples from adenoma's andcolorectal cancers is shown in Table 29. A specificity of 100% wasobtained for most of the genes, except for SFRP2. The best performinggenes in fecal samples from patients with CRC corresponded to GATA4 with73% sensitivity, SFRP1 with 67% sensitivity, OSMR3 with 67% sensitivity,and NDRG4 with 60% sensitivity, all with a corresponding specificity of100%.

TABLE 29 Performance of the markers in fecal samples SFRP OSMR NumberAct 2 GATA5 GATA4 APC SFRP1 NDRG4 (3) Adam23 cutoff of 200 1 1 4 1 1 010 1 (copies) samples Sens 13 15% 38%   0%  15%  8%  8%  15%  8%  0%adenoma   Sens CRC 15 67% 67%  27%  73%  47%  67%  60%  67%  40% Spec 1995% 84% 100% 100% 100% 100% 100% 100% 100%Performance of Marker Combination Panels in Fecal Samples

Four candidate methylation markers were found to result in the bestsensitivity and specificity in stool samples: GATA4, SFRP2, NDRG4, OSMR.β-Actin copy numbers were also quantified as a control for samplequality and DNA yield. The performance of combination panels of these 4methylation markers was investigated. Methylated copies of these geneswere quantified in all available stool samples by real-time MSP on a7900HT fast real-time PCR system (Applied Biosystems). Table 30 showsthe results and lists the cut-off (copies) applied. For instance for themost sensitive marker combination panel SFRP2+GATA4+NDRG4+OSMR, cutoffvalues of the individual markers were SFRP2=2; GATA4=4; NDRG4=0.1 andOSMR=10. This combination panel had 95% specificity, 87% sensitivity forCRC, and 46% sensitivity for adenomas. The preferred 2-markercombination NDRG4+GATA4 had a 100% specificity, a sensitivity of 73% forCRC, and a 33% sensitivity for adenomas.

TABLE 30 Performance of marker combinations Cutoff (copies) andperformance of combination panels * SFRP2 + SFRP2 + GATA4 + SFRP2 +GATA4 + SFRP2 + NDRG4 + NDRG4 + NDRG4 + NDRG4 + NDRG4 NDRG4 OSMR (3)OSMR (3) OSMR (3) GATA4 SFRP 1 1 1 1 (—) (—) 2 cp GATA4 4 (—) 4 (—) (—)4 cp NDRG4 0 0  0  0  0 0 cp OSMR (—) (—) 10 10 10 (—) (3) Sens 46% 46%46% 46% 23%  33% adenoma Sens 80% 73% 87% 80% 80%  73% CRC Speci- 95%95% 95% 95% 95% 100% ficity * Marker not used in the combination panelis indicated by (—)

The performance of the most sensitive marker combination panelSFRP2+GATA4+NDRG4+OSMR was evaluated for the different UICC stages.Results are summarized in Table 31.

TABLE 31 Performance of combination panel SFRP2 + GATA4 + NDRG4 + OSMRfor different UICC stages UICC stage Neg Pos Total ? 1 1 I 1 4 5 II 4 4III 1 3 4 IV 1 1 Total 2 13 15 samples

Example 2

Based on re-expression, 224 different gene assays representing 145 genepromotors were tested on the Base5 methylation profiling platform (datanot shown, see reference 2 for details). The 37 most differentiallymethylated gene sequences assessing 29 gene promoters were validated onretrospectively collected tumors from 65 colorectal cancer patients (allstages) and 74 distant resection ends (histopathologically normal) usingreal-time MSP. Several markers reliably detected CRC in those tissuesamples (data not shown). The results were confirmed on an independenttest set containing 59 samples from patients with cancer other than CRC(20 breast, 21 lung and 22 bladder cancer samples covering stagesI-III), 39 non-cancerous controls, 34 carcinomas and 16 adenomas. Aftertesting the non-CRC tissue samples, we had 59 results because 4 wereinvalid. The individual performance of the 9 best performing tissuemarkers is shown in FIG. 2, when the analytical cut-off was set to give100% specificity (based on the 39 non-cancerous controls). The mosttissue specific markers include: NDRG4, OSMR, SFRP1, ADAM23, GATA5 andMGMT.

REFERENCES

-   Ahlquist D A, Skoletsky J E, Boynton K A, Harrington J J, Mahoney D    W, Pierceall W E, Shuber A P. Colorectal cancer screening by    detection of altered human DNA in stool: feasibility of a    multi-target assay panel.-   Gastroenterology 2000, 119:1219-1227-   Baylin, S. B., Belinsky, S. A. & Herman, J. G. Aberrant methylation    of gene promoters in cancer-concepts, misconcepts, and promise.-   J. Natl Cancer Inst. 92, 1460-1461 (2000).-   Belshaw N J, Elliott G O, Williams E A, et al. Use of DNA from human    stools to detect aberrant CpG island methylation of genes implicated    in colorectal cancer.-   Cancer Epidemiol Biomarkers Prev 2004; 13:1495{circumflex over    ( )}501.-   Boynton K A, Summerhayes I C, Ahlquist D A, Shuber A P. DNA    integrity as a potential marker for stool-based detection of    colorectal cancer.-   Clin Chem 2003, 49:1058-1065-   W. D. Chen, Z. J. Han, J. Skoletsky, J. Olson, J. Sah, L.    Myeroff, P. Platzer, S. Lu, D. Dawson, J. Willis, T. P. Pretlow, J.    Lutterbaugh, L. Kasturi, J. K. Willson, J. S. Rao, A. Shuber    and S. D. Markowitz. Detection in fecal DNA of colon cancer specific    methylation of the nonexpressed vimentin gene. J Natl Cancer Inst 97    (2005), 1124-1132.-   Dong S M, Traverso G, Johnson C, Geng L, Favis R, Boynton K, Hibi K,    Goodman S N, D'Allessio M, Paty P, Hamilton S R, Sidransky D, Barany    F, Levin B, Shuber A, Kinzler K W, Vogelstein B, Jen J. Detecting    colorectal cancer in stool with the use of multiple genetic targets.    J Natl Cancer Inst. 2001 Jun. 6; 93(11):858-65-   P. A. Jones and S. B. Baylin. The fundamental role of epigenetic    events in cancer. Nat Rev Genet 3 (2002), 415-428.-   P. W. Laird. Early detection: The power and the promise of DNA    methylation markers. Nat Rev Cancer 3 (2003), 253-266.-   K. Lenhard, G. T. Bommer, S. Asutay, R. Schauer, T. Brabletz, B.    Goke, R. Lamerz and F. T. Kolligs. Analysis of promoter methylation    in stool: a novel method for the detection of colorectal cancer,    Clin Gastroenterol Hepatol 3 (2005), 142-149.-   Leung W K, To K F, Man E P, Chan M W, Bai A H, Hui A J, Chan F K,    Lee J F, Sung J J. Detection of epigenetic changes in fecal DNA as a    molecular screening test for colorectal cancer: a feasibility study.-   Clin Chem. 2004 November; 50(11):2179-82.-   H. M. Muller, M. Oberwalder, H. Fiegl, M. Morandell, G. Goebel, M.    Zitt, M. Muhlthaler, D. Ofner, R. Margreiter and M. Widschwendter.    Methylation changes in faecal DNA: a marker for colorectal cancer    screening? Lancet 363 (2004), 1283-1285.-   Olson J, Whitney D H, Durkee K, Shuber A P. DNA Stabilization Is    Critical for Maximizing Performance of Fecal DNA-Based Colorectal    Cancer Tests Diagn Mol Pathol. 2005 September; 14(3):183-91.-   Z. Petko, M. Ghiassi, A. Shuber, J. Gorham, W. Smalley, M. K.    Washington, S. Schultenover, S. Gautam, S. D. Markowitz and W. M.    Grady. Aberrantly methylated CDKN2A, MGMT, and MLH1 in colon polyps    and in fecal DNA from patients with colorectal polyps.-   Clin Cancer Res 11 (2005), 1203-1209.-   Sidransky, D. Nucleic acid-based methods for the detection of    cancer. Science 278, 1054-1058 (1997)-   Straub, J. et al., AB-104-AACRMD (2007), poster presented September    2007 at the AACR meeting “Molecular Diagnostics in Cancer    Therapeutic Development: Maximizing Opportunities for Personalized    Treatment.-   Whitney D, Skoletsky J, Moore K, Boynton K, Kann L, Brand R, Syngal    S, Lawson M, Shuber A. Enhanced Retrieval of DNA from Human Fecal    Samples Results in Improved Performance of Colorectal Cancer    Screening Test. J Mol Diagn. 2004 November; 6(4):386-95-   Zou et al., Clin Chem. 2007 September; 53(9):1646-51. A novel method    to capture methylated human DNA from stool: implications for    colorectal cancer screening.    3) Experiments on Plasma DNA    Materials and Methods in Relation to Plasma DNA    Sample Collection and Processing

Plasma samples were collected from multiple centers in Germany, TheNetherlands and Belgium.

10 ml of blood was obtained per individual using EDTA Vacutainer™ tubes.Individuals with no suspicious findings, adenomas or carcinomas based oncolonoscopy were enrolled in the present study. Within 4 hrs from theblood drawing, the plasma fraction was separated from the cell fractionby centrifugation at 1500 g for 15 min (4° C.). The plasma wastransferred to new tubes and once again centrifuged (1500 μg, 15 min, 4°C.), after which the supernatant was transferred to new tubes and storedat −80° C. until further use. Samples were shipped on dry ice.

Plasma samples from patients with stages I-IV of colorectal cancers anddifferent controls belonging to the following groups were enrolled inthis study. Tables 32 and 33 gives an overview of the collected samplessets.

-   -   Colorectal cancer group: patients with pathologically confirmed        colorectal cancer with stage I to IV (according to the UICC        stage grouping)    -   Adenomas    -   Non-cancer controls: patients without cancerous disease    -   Cancer controls: patients with carcinomas other than colorectal        cancer

TABLE 32 Plasma training set 1 Diagnosis Sample Number of group volumesamples Notes Colorectal cancers 1.2 to 4.5 ml of 42 StageI-IV plasmaGrade 1-3 (corresponding to (81% stage I-III) Non-cancer 0.07 to 0.27plasma 34 Symptomatic controls equivalent of DNA patients with non- perPCR) acute conditions Cancer controls 4 to 6 ml of 25 Predominantlyplasma ovarian and (corresponding to prostate cancers 0.24 to 0.36plasma equivalent of DNA per PCR)

TABLE 33 Plasma training set 2 Diagnosis Sample Number of group volumesamples Notes Colorectal cancers 1.3 to 4.3 ml of 78 StageI-IV plasmaGrade 1-3 (corresponding to (76% stage I-III) Adenomas 0.16 to 0.52 49Non-cancer controls plasma equivalent 64 Symptomatic of DNA per PCR)patients with non-acute conditions Cancer controls 4 to 6 ml of 25Predominantly plasma ovarian and prostate (corresponding to cancers 0.48to 0.72 plasma equivalent of DNA per PCR)DNA Isolation from Plasma Samples

DNA isolation from plasma samples (1.2 to 6 ml) was performed using anupscaled phenol-chloroform DNA isolation method using the 15 ml of HeavyPhase lock Gel tubes (PLG tubes) (Eppendorf, cat #0032 005.152) oralternatively the ChargeSwitch® gDNA 1 ml serum kit from Invitrogen (cat#CS11040).

Phenol-Chloroform Procedure

Plasma samples were thawed and 1/10 volume of 10× buffer (240 mM EDTA(pH=8.0), 750 mM NaCl), 1/10 volume of 10% SDS and 5 μl of Proteinase K(20 mg/ml stock solution) per 1 ml of sample (e.g. 15 μl for 3 ml ofsample) was added to each plasma sample. This mixture was incubatedovernight at 48° C. at constant shaking (200 RPM).

Subsequently the PLG tube was centrifuged at 2500 RCF for 3 min, samplemixture and approximately the same volume of phenol/chloroform(Invitrogen, cat #15593049) were added to it. This solution was brieflyvortexed, mixed for 10 min using a tube rocker at room temperature andcentrifuged for 5 min at 2500 RCF. In case the retrieved sample volumewas ≤4 ml, an equal volume of phenol/chloroform was added. The upperaqueous layer was phenol/chloroform-treated for a second time.

DNA was precipated from the the upper aqueous layer by adding 5 μlglycogen, 1/10 volume of 7.5 M Ammonium Acetate and 2-2.5 volumes ofcold (−20° C.) 100% ethanol. Tubes were gently inverted and incubated at−20° C. for at least 1 h, followed by a centrifugation step at 17000 RCFfor 30 min (4° C.). Ethanol was carefully removed by pipetting. Pelletswere washed with 2 ml freshly prepared 70% ethanol, vortexed gently andsubmitted to a centrifugation step at 17000 RCF for 15 min at 4° C.After careful removal of the remaining ethanol, pellets were air driedand resuspended in 45 μl of LoTE pH 8.0. The isolated DNA is stored at−80° C. until further processing. This method allowed an average DNArecovery of 120 ng per ml of plasma.

ChargeSwitch® gDNA 1 ml Serum Kit

Plasma samples are thawed and DNA is isolated using the ChargeSwitch®gDNA 1 ml serum kit according to the manufacturer's instructions withthe exception that the procedure is upscaled for larger sample volumesusing the MagnaBot® large volume magnetic separation device from Promega(Cat #V3471). Results are presented in Table 41.

DNA Modification

The complete content of DNA isolated in above procedure was subjected tosodium bisulfite treatment (BT) using the EZ-96 DNA Methylation kit fromZymo Research (Cat #D5003) performed on a pipetting robot (Tecan FreedomEVOII, Roma, Liha, Mca, Te-Vacs). Briefly, 45 μl of plasma DNA samplewas mixed with 5 μl of M-Dilution Buffer (provided in kit) and incubatedat 37° C. for 15 min shaking at 1100 RPM. This mixture was furtherincubated with 100 μl of diluted CT conversion reagent (provided in kit)shaking at 70° C. for 3 hours (protected from light). Subsequently themodified DNA was desalted and desulfonated according to manufacturer'sinstructions and eluted in either 40 μl or 20 μl of Tris-HCl 1 mM pH8.0,depending on the applied concentration procedure. The eluted materialwas stored at −80° C. until further processing.

DNA Amplification

Real-time MSP was performed on a 7900HT fast real-time PCR cycler fromApplied Biosystems. 2.4 μl of the modified DNA was added to a PCR mix(total volume 12 μl) containing home-made buffer solution (finalconcentrations are summarized: 16.6 mM (NH₄)₂SO₄, 67 mM Tris (pH 8.8),6.7 mM MgCl2, 10 mM β-mercaptoethanol), dNTPs (5 mM; AmershamBiosciences cat #27-2035-02), methylation specific forward primer (6ng), methylation specific reverse primer (18 ng), molecular beacon (0.16μM) and Jumpstart DNA Taq polymerase (0.4 units; Sigma Cat #D9307).

Cycling conditions are specified in Table 34.

A standard curve was included (9.6×10⁵−9.6 copies) to determine copynumbers of unknown samples by interpolation of their Ct values to thestandard curve.

TABLE 34 Cycling profile 1 Activation 95° C.  5 min 2 Denaturation 95°C. 30 sec 3 Annealing and 57° C. 30 sec data (51° C. for APC) collection4 extension 72° C. 30 sec 5 cycling Repeat step 2 to 4, 45 timesResultsMarker Identification and Validation in Tissue and Plasma Samples.Assay Validity Rate in Tissue and Plasma:

293 FFPE and 317 plasma samples were processed using real-time MSP(Table 35). The real-time MSP assays produced valid results in 98% ofthe FFPE samples and in 100% of the plasma samples.

TABLE 35 Summary of samples evaluated by real-time MSP Sample SampleSets Sample Types Numbers Valid Tests [%] Tissue Cancer 65 65/65 [100]Training Set Controls 76 74/76 [97] Total 141 139/141 [99] Tissue TestCRC 34 34/34 [100] Set Controls 39 39/39 [100] Other Cancers 63 59/63[94] Adenomas 16 16/16 [100] Total 152 148/152 [97] Tissue Sets CRC 9999/99 [100] combined Controls 115 113/115 [98] Other Cancers 63 59/63[94] Adenomas 16 16/16 [100] Total 293 287/293 [98] Plasma Cancer 4242/42 [100] Training set Controls 34 34/34 [100] (1) Other cancers 2525/25 [100] Total 101 101/101 [100] Plasma Cancer 78 78/78 [100]Training set Adenoma 49 49/49 [100] (2), Controls 64 64/64 [100]increased Other cancers 25 25/25 [100] plasma Total 216 216/216 [100]equivalent of DNA per real-time MSP assay Plasma Sets Cancer 120 120/120[100] combined Adenoma 49 49/49 [100] Controls 98 98/98 [100] Othercancers 50 50/50 [100] Total 317 317/317 [100]Marker Identification

Using re-expression profiles of colon cancerous cell lines, candidategenes were identified and the most promising markers (224 different geneassays representing 145 gene promoters) were tested on tissue using theBaseS methylation profiling platform (data not shown, see Straub, J. etal for details). Promoter sequences were linked with gene expression toidentify epigenetically silenced genes. An established pharmacologicunmasking strategy (5-aza-2′-deoxycytidine (DAC) and trichostatin A(TSA)) for re-expression analysis of epigenetically targeted genes wascombined with proprietary advanced bioinformatics tools to identifygenes prone to promoter methylation.

Marker Selection in Colon Tissue

Marker candidates identified by re-expression were screened using 37real-time methylation specific PCR (real-time MSP) assays. These assayswere used to assess the methylation status of 29 gene promoters in 293formalin-fixed paraffin-embedded (FFPE) tissue samples collected fromvarious clinics. Samples included 99 carcinomas of various stages, 16adenomas, 63 samples from patients with cancer other than CRC (20 breast[stages I-III], 22 bladder [stages I-III], 21 lung [stages I and II]),39 samples from patients with no evidence of cancer and 76 distantresection ends (histopathologically normal) from CRC patients. Thesesamples were divided into training and independent test sets, and usedto select the gene methylation assays best able to discriminate betweencancerous and non-cancerous samples. The training set includedretrospectively collected tumors from 65 colorectal cancer patients (allstages) and 74 distant resection ends. Using the 10 best performinggenes the results were confirmed on an independent test set containing59 samples from patients with cancer other than CRC, 39 non-cancerouscontrols and 50 cancer cases (34 carcinomas and 16 adenomas). Theindividual performance of the 10 best performing tissue markers OSMR,SFRP1, GATA4, SFRP2, NDRG4, ADAM23, GATA5, MGMT, APC and JPH3 is shownin FIG. 7, when the analytical cut-off was set to give 100% specificity,except for JPH3 where a specificity of 95% was obtained (based on the 39non-cancerous controls). Corresponding primer and beacon sequences aresummarized in Table 3 (above). In addition to the colon test genes, theindependent reference gene β-Actin (ACT) was also measured. The ratiosbetween the colon test genes and ACT were calculated, and are the testresult of the assay. The samples were classified as methylated,non-methylated, or invalid based on the decision tree shown in FIG. 5.

Complementarity of Markers

The different markers were tested on their complementarity. Severalmarker combinations reliably detected CRC with high specificity andsensitivity. Results of the best 2 marker combinations are summarized inTable 36. For 100% specificity, sensitivities ranged between 94 to 100%.

TABLE 36 Performance of 2 combinations of the markers reliably detectingCRC and adenomas when using real-time MSP (tissue test set: 34carcinomas, 16 adenomas, 39 controls) Specificity % # detected/Sensitivity % (# detected/ Samples tested [95% CI] tested) Panel 1(OSMR, GATA4, ADAM23) 34 carcinomas 33/34 97 [91-100] 100 (0/39) 39controls 16 adenomas 16/16 100 100 (0/39) 39 controls 50 neoplasms 49/5098 [94-100] 100 (0/39) (34 carcinomas and 16 adenomas) 39 controls Panel2 (OSMR, GATA4, GATA5) 34 carcinomas 32/34 94 [86-100] 100 (0/39) 39controls 16 adenomas 16/16 100 100 (0/39) 39 controls 50 neoplasms 48/5096 [90-100] 100 (0/39) (34 carcinomas and 16 adenomas) 39 controlsMarker Testing in Plasma

Eight of the best performing markers in tissue were assessed (OSMR,SFRP1, NDRG4, GATA5, ADAM23, JPH3, SFRP2 and APC) on 101 availableplasma samples from multiple centers (plasma training set 1: Table 32).These plasma samples included 34 samples with no suspicious findings, 25samples from patients with cancers other than colon cancer and 42samples from patients covering all stages of CRC, with 81% representingstages I-III of disease.

DNA was isolated following the upscaled phenol-chloroform procedure;subsequently the whole DNA sample was modified as described above. Theplasma training set 1 was eluted in 40 μl of BT elution volume of which2.4 μl was subjected to real-time MSP, the 2.4 μl of eluted DNAcorresponds to an equivalent of 0.07 to 0.36 ml of original plasmasample which went into the isolation procedure (=0.07 to 0.36 plasmaequivalent of DNA per PCR).

The individual performance (% sensitivity) of the 8 gene assays inplasma samples is shown in FIG. 8, sensitivity values ranging from 14 to33%. Corresponding specificity values are displayed in Table 37.Obtained specificity values ranged from 97 to 100%.

Five of the best performing markers in training set 1 were furtherstudied with an additional, independent sample set prospectivelycollected from multiple centers (plasma training set 2: Table 33).Reducing the number of gene assays from 8 to 5 resulted in fewer assaysper sample and a greater aliquot of plasma equivalent of DNA was addedper PCR reaction. The modified DNA from sample set 2 was moreconcentrated by eluting in 20 μl instead of 40 μl of BT elution volume.2.4 μl eluted DNA from sample set 2 was further processed throughreal-time MSP, this corresponds to 0.16 to 0.72 ml plasma equivalent ofDNA per PCR depending on the plasma volume prior to DNA isolation.

The plasma samples of training set 2 included 64 samples with nosuspicious findings, 49 adenomas, 25 samples from patients with cancersother than colon cancer and 78 samples from patients covering all stagesof CRC, with 76% representing stages I-III of disease. The individualperformance (% sensitivity) of the 5 gene assays is shown in FIG. 8 withcorresponding specificity values displayed in Table 37. Specificityvalues ranged from 96 to 99%, with sensitivity ranging from 23 to 47%.

Four candidate methylation markers were found to result in the bestsensitivity and specificity in plasma samples: OSMR, NDRG4, GATA5 andADAM23; performance of this plasma panel is shown in Table 38.Performance characteristics (stages I-III CRC) of this panel of 4methylation genes demonstrated 73% sensitivity and 92% specificity whenoptimized for sensitivity, whereas 64% sensitivity and 98% specificitywas obtained when optimizing for specificity. Sensitivity can be furtherimproved (from 64% to 68%) when samples with a plasma volume less than 2ml prior to DNA isolation are excluded from analysis. Results arepresented in Table 39.

TABLE 37 Individual gene assay performance displaying % specificity forboth plasma training sets and % sensitivity for adenomas in plasmatraining set 2 OSMR SFRP1 NDRG4 GATA5 ADAM23 JPH3 SFRP2 APC %Specificity 100 98 100 97 98 97 97 97 (all 59 controls), plasma set 1 %Specificity 99 96 99 99 97 N/A N/A N/A (all 89 controls), plasma set 2:increased plasma equivalent of DNA per real- time MSP assay %Sensitivity 2 2 0 6 2 N/A N/A N/A adenomas plasma set 2: increasedplasma equivalent of DNA per real- time MSP assay

TABLE 38 Performance of a plasma marker panel using real-time MSP(independent of recovered plasma volume prior to DNA isolation)Specificity % Sensitivity % (# detected/# Sample (# detected/# total)total) sets Sample groups [95% CI] [95% CI] Plasma panel (optimized forsensitivity) OSMR, NDRG4, GATA5 and ADAM23 Plasma Stages I-III CRC 50(17/34) 97 (2/59) [93-100] training All Stages CRC 60 (25/42) [45-83]set 1 All Controls Plasma Stages I-III CRC 73 (43/59) 92 (7/89) [86-98]training All Stages CRC 73 (57/78) [63-83] set 2 Adenomas 12 (6/49)(increased All Controls plasma equivalent of DNA per real- time MSPassay) Plasma panel (optimized for specificity) OSMR, NDRG4, GATA5 andADAM23 Plasma Stages I-III CRC N/A N/A training All Stages CRC set 1 AllControls Plasma Stages I-III CRC 64 (38/59) 98 (2/89) [95-100] trainingAll Stages CRC 64 (50/78) [53-75] set 2 Adenomas  6 (3/49) (increasedAll Controls plasma equivalent of DNA per real- time MSP assay)

TABLE 39 Performance of a plasma marker panel using real-time MSP usingat least 2 ml of plasma prior to DNA isolation Sensitivity % Specificity% (# detected/# (# detected/# total) total) Sample sets Sample groups[95% CI] [95% CI] Plasma panel (optimized for sensitivity) OSMR, NDRG4,GATA5 and ADAM23 Plasma Stages I-III 73 (41/56) 92 (7/89) training set 2CRC 74 (54/73) [64-84] [86-98] (increased All Stages CRC 12 (6/49)plasma Adenomas equivalent of All Controls DNA per real- time MSP assay)Plasma panel (optimized for specificity) OSMR, NDRG4, GATA5 and ADAM23Plasma Stages I-III 68 (38/56) 98 (2/89) training set 2 CRC 67 (49/73)[56-76] [95-100] (increased All Stages CRC  6 (3/49) plasma Adenomasequivalent of All Controls DNA per real- time MSP assay)Average DNA Recovery Yield from Plasma Samples

Plasma DNA (collected after double centrifugation step) from colorectalcancer patients was isolated according to the phenol/chloroformprocedure and quantified using the PicoGreen dsDNA quantitation kit fromMolecular Probes. The average plasma DNA recovery yield was 117 ng/ml ofplasma, with a range of 41 to 384 ng/ml (data obtained from 25patients).

TABLE 40 Average DNA recovery yield plasma samples ng/ml Sample plasma 141 2 66 3 264 4 163 5 54 6 121 7 87 8 107 9 53 10 121 11 88 12 201 13 5314 47 15 384 16 87 17 115 18 107 19 70 20 72 21 122 22 146 23 71 24 19525 94Phenol/Chloroform Procedure Versus ChargeSwitch® Using Plasma Samples

This experiment was carried out to show the isolation of DNA from plasmaby using the method of this invention. Plasma volumes ranging from 2.5to 6 ml were processed according to the above discussed upscaledphenol/chloroform and ChargeSwitch® isolation procedure. Plasma derivedfrom ovarian, prostate and colon blood samples were investigated. Theobjective was to isolate DNA (according to both methods) and furtherprocess the samples in parallel through bisuphite treatment and β-Actinreal-time MSP to address the sample quality and DNA yield. Thecorresponding β-Actin copies for both isolation procedures aresummarized in Table 16.

TABLE 41 β-Actin copies phenol/chloroform versus ChargeSwitch ®isolation procedure B-Actin B-Actin Sample Sample Plasma copies copiesnumber origin volume (ml) Phenol ChargeSwitch 1 ovarian 6.0 4349 863cancer 2 ovarian 6.0 2710 466 cancer 3 ovarian 6.0 3922 967 cancer 4ovarian 6.0 758 490 cancer 5 ovarian 6.0 4201 423 cancer 6 ovarian 6.02644 139 cancer 7 ovarian 6.0 1472 187 cancer 8 prostate 2.6 145 7cancer 9 colon 2.5 317 52 cancer 10 pos control N/A 8702 1314 cell lineUpdated Results for Plasma Training Set 2.

Corrected information was received from the clinics about plasmatraining set 2. For plasma training set 2: the cancer cases remained thesame, a new category of “unknown” was created, the number of controlswas 52 (instead of former 64) and the adenoma cases were 39 (instead offormer 49). This allowed re-classification of sample types as providedin Table 42. Since the corrected information classified a number ofunknown cancer cases (controls) as early stage cancers, additionalconclusions on detection of early stage cancers could be drawn. As shownin table 43, the plasma panel allowed very sensitive detection (70%) ofearly stage samples. Improved detection could be obtained by excludingsamples with a plasma volume less than 2 ml (Table 44)

TABLE 42 Summary of samples tested by real-time MSP and evaluablilityrate Sample Sample sets Sample types numbers Valid tests [%] Tissue CRC65 65/65 [100] training set Controls 76 74/76 [97] Total 141 139/141[99] Tissue test CRC 34 34/34 [100] set Controls 39 39/39 [100] OtherCancers 63 59/63 [94] Adenomas 16 16/16 [100] Total 152 148/152 [97]Tissue sets CRC 99 99/99 [100] combined Controls 115 113/115 [98] OtherCancers 63 59/63 [94] Adenomas 16 16/16 [100] Total 293 287/293 [98]Plasma CRC 42 42/42 [100] training set Controls 34 34/34 [100] (1) Othercancers 25 25/25 [100] Total 101 101/101 [100] Plasma CRC 78 78/78 [100]training set Adenoma 39 49/49 [100] (2) Controls 52 64/64 [100] Othercancers 25 25/25 [100] Unknown 22 22/22 [100] Total 216 216/216 [100]Plasma sets CRC 120 120/120 [100] combined Adenoma 39 49/49 [100]Controls 86 98/98 [100] Other cancers 50 50/50 [100] Unknown 22 22/22[100] Total 317 317/317 [100]

TABLE 43 Performance characteristics of a 4-gene marker panel usingplasma set 2 Sample Plasma panel OSMR, GATA5, NDRG4 and ADAM23 groupsoptimized for sensitivity optimized for specificity (plasma Sensitivity% Specificity % Sensitivity % Specificity % training (# detected/#total) (# detected/# total) (# detected/# total) (# detected/# total)set 2) [95% CI] [95% CI] [95% CI] [95% CI] Early 70% (23/33) 92% (6/77)58% (19/33) 99% (1/77) stages CRC [54-86] [86-98] [41-75] [96-100](0-II) All stages 73% (57/78) 64% (50/78) CRC [63-83] [53-75] Adenomas10% (4/39)  5% (2/39) Controls

TABLE 44 Performance characteristics of a 4-gene marker panel usingplasma set 2 Sample Plasma panel OSMR, GATA5, NDRG4 and ADAM23 groupsoptimized for sensitivity optimized for specificity (plasma Sensitivity% Specificity % Sensitivity % Specificity % training (# detected/#total) (# detected/# total) (# detected/# total) (# detected/# total)set 2) [95% CI] [95% CI] [95% CI] [95% CI] Early 70% (23/33) 92% (6/77)58% (19/33) 99% (1/77) stages CRC [54-86] [86-98] [41-75] [96-100](0-II) All stages 74% (54/73) 67% (49/73) CRC [64-84] [56-78] Adenomas10% (4/39)  5% (2/39) Controls

REFERENCES

-   Baylin, S. B., Belinsky, S. A. & Herman, J. G. Aberrant methylation    of gene promoters in cancer—concepts, misconcepts, and promise. J.    Natl Cancer Inst. 92, 1460-1461 (2000).-   Catherine Lofton-Day et al, poster presented April 2007 at the AACR    Annual meeting 2007, Los Angelos, USA: “Clinical case-control study    in plasma shows that the DNA methylation biomarker, Septin 9,    detects 70% of Stage I-III colorectal cancer patients”-   W. M. Grady, A. Rajput, J. D. Lutterbaugh and S. D. Markowitz,    Detection of aberrantly methylated hMLH1 promoterDNA in the serum of    patients with microsatellite unstable colon cancer, Cancer Res 61    (2001), 900-902-   P. A. Jones and S. B. Baylin. The fundamental role of epigenetic    events in cancer. Nat Rev Genet 3 (2002), 415-428.-   P. W. Laird. Early detection: The power and the promise of DNA    methylation markers. Nat Rev Cancer 3 (2003), 253-266.-   Leung W K, To K F, Man E P, Chan M W, Bai A H, Hui A J, Chan F K,    Sung J J. Quantitative detection of promoter hypermethylation in    multiple genes in the serum of patients with colorectal cancer. Am J    Gastroenterol. 2005 October; 100(10):2274-9-   Nakayama G, Hibi K, Nakayama H, Kodera Y, Ito K, Akiyama S, Nakao A.    A highly sensitive method for the detection of p16 methylation in    the serum of colorectal cancer patients. Anticancer Res. 2007    May-June; 27(3B):1459-63-   Straub, J. et al., AB-104-AACRMD (2007), poster presented September    2007 at the AACR meeting “Molecular Diagnostics in Cancer    Therapeutic Development: Maximizing Opportunities for Personalized    Treatment.-   Yamaguchi S, Asao T, Nakamura J, Ide M, Kuwano H.-   High frequency of DAP-kinase gene promoter methylation in colorectal    cancer specimens and its identification in serum.-   Cancer Letters, 2003 May 8; 194(1): 99-105-   Hong-Zhi Zou, Bao-Ming Yu2, Zhi-Wei Wang, Ji-Yuan Sun, Hui Cang, Fei    Gao, Dong Hua Li, Ren Zhao, Guo-Guang Feng and Jing Yi. Detection of    aberrant p16 methylation in the serum of colorectal cancer patients.    Clin Cancer Res. Vol. 8, 188-191, January 2002.    4) N-Myc Downstream Regulated Gene 4 (NDRG4) Promoter Methylation is    a Sensitive and Specific Biomarker for Colorectal Cancer    Abstract

Background and aims: N-Myc downstream regulated gene 4 (NDRG4), a geneinvolved in cellular differentiation and neurite formation, is one ofthe four members of the NDRG family. Here we address the role of NDRG4promoter methylation in CRC (CRC).

Methods: NDRG4 promoter methylation was analyzed in CRC cell lines, wellcharacterised series of normal colon mucosa, colorectal adenomas,carcinomas and other neoplasias using methylation specific PCR (MSP) andbisulfite sequencing. NDRG4 promoter methylation was also analyzed infecal DNA of CRC patients and controls using quantitative MSP. Loss ofheterozygosity (LOH) mapping of the NDRG4 locus and mutation analysisusing direct sequencing of NDRG4 coding exons and their flankingintronic regions were performed. NDRG4 mRNA and protein expression wasstudied using RT-PCR and immunohistochemistry respectively.

Results: NDRG4 promoter methylation is observed in 7/8 CRC cell lines.The prevalence of NDRG4 promoter methylation in CRC tissue is 86%(71/83) compared to 4% (2/48) in normal colon mucosa. A second,independent series of CRCs confirmed the high prevalence (69%, 127/183)of NDRG4 methylation. NDRG4 methylation was also observed in 81% (13/16)of oesophageal adenocarcinomas and 77% (17/22) of gastric cancers whileno or little methylation was observed in skin (0/8), kidney (1/10),ovary (0/20), prostate (0/10), breast (0/16) and oesophageal squamouscell cancers (0/12). NDRG4 promoter methylation can be detected in fecalDNA of 76% (16/21) of CRC patients, while only 3% (2/67) of controlpatients tested positive yielding a sensitivity of 76% and a specificityof 97%. No mutations were found and 30.5% of tumors showed LOH on theNDRG4 locus. Expression of NDRG4 is decreased at the RNA and proteinlevel in CRC when compared to normal tissue.

Conclusions: NDRG4 is frequently methylated in CRC cell lines,colorectal adenomas and carcinomas and other adenocarcinomas of thegastrointestinal tract. NDRG4 promoter methylation in fecal DNA can beused as a sensitive and specific biomarker for the detection of CRC.

Introduction

Previous microarray experiments to identify genes which areepigenetically regulated in tumor endothelial cells revealed 81 genesthat are downregulated in tumor endothelial cells and reexpressed after5-aza-2′-deoxycytidine (DAC) and trichostatin A (TSA) treatment.Silencing of these genes in tumor-endothelial cells was associated withpromoter histone H3 deacetylation and loss of H3 lysine 4 methylation,however did not involve DNA methylation of promoter CpG islands.Interestingly, 21 of these 81 genes (26%) have been reported to behypermethylated and silenced in various tumor types suggesting that manyof the identified gene promoters have the potential to be regulated bypromoter methylation in tumor cells (Hellebrekers, Melotte et al. 2007).Amongst the identified CpG island containing genes is N-mycdownregulated gene-4 (NDRG4), also known as Smap-8 and Bdm1. NDRG4 ispart of the NDRG family which consists of four members, NDRG1, -2, -3and -4 which have an amino acid sequence homology of 57-65% (Zhou,Kokame et al. 2001; Qu, Zhai et al. 2002). Phylogenetic analysisverified two subfamilies, one consisting of NDRG1 and -3 and the otherconsisting of NDRG-2 and -4 (Qu, Zhai et al. 2002). NDRG1 is the mostextensively studied member of the NDRG family. Expression of NDRG1 isoften downregulated in cancer cells (van Belzen, Dinjens et al. 1997;Kurdistani, Arizti et al. 1998; Guan, Ford et al. 2000; Bandyopadhyay,Pai et al. 2003; Bandyopadhyay, Pai et al. 2004; Shah, Kemeny et al.2005) and upregulated by DAC treatment (Guan, Ford et al. 2000;Bandyopadhyay, Pai et al. 2004). In addition, NDRG2 has also beendescribed as candidate tumor suppressor gene (Deng, Yao et al. 2003;Lusis, Watson et al. 2005) and reported to be methylated in meningiomas(Lusis, Watson et al. 2005) and different cancer cell lines (Liu, Wanget al. 2007). So far, the function of NDRG3 and NDRG4 in cancer has notbeen addressed. The NDRG4 gene is located on chromosome 16q21-q22.3,spans 26 kb and contains 17 exons covering the entire sequence of threecDNA isoforms NDRG4-B, NDRG4-Bvar and NDRG4-H. NDRG4 mRNA ispredominantly present in the cytoplasm. At present, expression of NDRG4has only been described in brain and heart using Northern blot analysis.The molecular characterization of NDRG4 and the role of this protein inthe nervous system has mainly been investigated in the rat (Nakada,Hongo et al. 2002; Ohki, Hongo et al. 2002; Maeda, Hongo et al. 2004;Hongo, Watanabe et al. 2006). NDRG4 protein may participate in processesthat lead to cellular differentiation and neurite formation (Ohki, Hongoet al. 2002).

Here, we report NDRG4 to be expressed in normal colon mucosa anddownregulated in colon cancer tissue. In addition, NDRG4 promotermethylation, loss of heterozygosity (LOH) and mutational inactivationwere examined. We identified the NDRG4 promoter as being frequentlymethylated in CRC and other neoplasias of the gastrointestinal tract andinvestigated its potential as a biomarker in stool of CRC patients andcontrols.

Materials and Methods

Cell Lines, Study Population and Tissues

CRC cell lines HT29, SW480, Caco2, Colo205, RKO, LS174T, HCT116 andSW480 were cultured in DMEM (Invitrogen) supplemented with 10%heat-inactivated fetal calf serum (Hyclone). To investigate reexpressionof NDRG4 following inhibition of DNA methyltransferases, HCT116 and RKOwere treated with 1 μM DAC (Sigma).

NDRG4 promoter methylation was investigated in well-characterized seriesof colorectal carcinomas, adenomas and controls (FIG. 9). The firstseries consists of formalin-fixed, paraffin-embedded CRCs (n=90) ofpatients over 50 years of age which were retrospectively collected fromthe archive of the dept. of Pathology of the University HospitalMaastricht. When present, also normal (n=79) and adenoma (n=60) tissuewas collected from these patients. Histologically normal biopsy materialfrom patients undergoing endoscopy for non-specific abdominal complaints(n=51), adenoma biopsies (n=22) from patients who did not develop CRCwithin 10 years, and resected colon mucosa of patients with variousinflammatory bowel conditions (n=33) were selected as control tissue.This last group includes Crohn's disease (n=1), colitis ulcerosa (n=6),non-specific inflammation (n=9) and diverticulitis (n=18). A secondindependent series of CRCs (n=200) was randomly selected from theprospective Netherlands Cohort Study on diet and cancer (NLCS), whichhas been described in detail elsewhere (van den Brandt, Goldbohm et al.1990; Brink, de Goeij et al. 2003). Series characteristics are shown insupplemental table 1 In addition, archival, formalin-fixed,paraffin-embedded skin—(n=8), kidney—(n=10), ovary—(n=10),prostate—(n=10), breast—(n=15), stomach—(n=22) and oesophagus (n=28)cancer tissue was analyzed for NDRG4 promoter methylation. This studywas approved by the Medical Ethical Committee (MEC) of the MaastrichtUniversity and the University Hospital Maastricht.

TABLE 45 Series characteristics Location^(‡) Age* Sex^(†) ProximalDistal CRC+ Normal tissue 71.0 ± 8.6 41/38 40/75 (53%) 35/75 (47%)Adenoma tissue 71.7 ± 7.9 32/30 26/59 (44%) 33/59 (56%) Carcinoma tissue71.5 ± 8.3 44/46 49/88 (56%) 39/88 (44%) CRC− Normal tissue 65.2 ± 9.022/29 13/39 (33%) 26/39 (67%) Adenoma tissue 63.1 ± 7.6 16/6  6/18 (33%)12/18 (67%) Inflamed tissue  65.3 ± 10.1 14/19 10/26 (39%) 16/26 (62%)P-value <0.001 NS NS CRC+ CRC− Carcinoma Adenoma Adenoma tissue tissuetissue Histological Histological type type Adenocarcinoma 72/90 (80%)39/62 (63%) 16/22 (73%) Mucinous 18/90 (20%) Tubular 22/62 (36%) 6/22(27%) carcinoma Tubulovillous 1/62 (2%) 0/22 (0%) VillousDifferentiation: Dysplasia Poor 8/90 (9%) Lowgrade 54/62 (87%) 22/22(100%) Moderate 70/90 (78%) Highgrade 8/62 (13%) 0/22 (0%) Well 12/90(13%) TNM stage: I 13/90 (14%) II 29/90 (32%) III 36/90 (40%) IV 12/90(13%) Table 4.5: Patient characteristics NDRG4b *years ± SD, analyzed byOne-way ANOVA ^(†)Male/Female, analyzed by Pearson's χ² ^(‡)analyzed byPearson's χ². Location could not be traced for all samples explainingdifferent total sample numbers CRC+: colorectal cancer patients CRC−:patients without colorectal cancer NS: not significant TNM stage:‘Tumour Node Metastasis’ StagingDNA-Isolation from Tissues and Cell Lines

A 5 μm section of each tissue block was stained with haematoxylin andeosin and revised by a pathologist (AdB). Five sections of 20 μm weredeparaffinated prior to DNA-isolation. DNA was extracted from thesetissue samples and from cell lines using the Puregene DNA isolation kit(Gentra systems) according to the manufacturers instructions. In brief,cell lysis solution and proteinase K (20 mg/ml, Qiagen) were added tothe tissue samples and incubated overnight at 55° C. Subsequently, DNAwas extracted for 72 h at 37° C., protein was removed, and DNA wasprecipitated using 100% 2-propanol. Finally, DNA was rehydrated inhydration buffer.

Collection and Preparation of Fecal DNA

Colonoscopy negative control stool samples (n=67) were obtained from apopulation of healthy subjects over 50 years of age which are beingscreened within the framework of a workplace-based community CRCscreening study at the University Hospital Maastricht. The MedicalEthical Committee (MEC) of the Maastricht University, the UniversityHospital Maastricht and the Dutch ‘Wet op Bevolkingsonderzoek’ (WBO) isapproving this screening study. Stool samples from colonoscopy confirmedCRC patients (n=21) covering all CRC stages were collected at the FreeUniversity Medical Center in Amsterdam. For recovery of human DNA, wholestool samples were homogenized in a 7 excess volume of stoolhomogenization buffer (Exact sciences, Marlborough, Mass., USA) andaliquoted in portions of 32 ml containing the equivalent of 4 g of stooleach. Single aliquots were centrifuged and the supernatants wereincubated with 80 units per ml RNase A for 60 minutes at 37° C. TotalDNA was then precipitated using sodium acetate isopropanol (PH 5.2),washed with 70% ethanol and resuspended in 4 ml 1×TE (pH 7.4). 400 μl10× buffer (240 mM EDTA (pH 8.0), 750 mM NaC), 400 μl 10% SDS and 20 μlProteinase K (20 mg/ml) was added, samples were incubated overnight at48° C. at constant shaking and centrifuged the next day. Additionally, 5ml of phenol-chloroform-isoamylalcohol was added and samples wereincubated for 10 minutes at RT before centrifugation. Thephenol-chloroform-isoamylalcohol extraction was repeated, the aqueouslayer was subsequently transferred in a new tube, DNA was precipitated,washed and pellets were resuspended in 2 ml of LoTE (pH 8.0).

Sodium Bisulfite Conversion, Methylation-Specific PCR and SodiumBisulfite Sequencing

Sodium bisulfite modification of 500 ng genomic DNA was performed usingthe EZ DNA methylation kit (ZYMO research Co., Orange, Calif.) accordingto the manufacturer's instructions. NDRG4 MSP analysis on bisulfitetreated DNA retrieved from cell lines and formalin-fixed, paraffinembedded tissue was facilitated by first amplifying the DNA withflanking PCR primers which amplify bisulfite-modified DNA but do notdiscriminate between methylated or unmethylated DNA. This PCR productwas used as a template for the MSP reaction (Herman, Graff et al. 1996;van Engeland, Weijenberg et al. 2003). Flank primers, MSP primers andPCR conditions are listed in table 2 (see above). All PCRs wereperformed with controls for unmethylated DNA (DNA from normallymphocytes), methylated DNA (normal lymphocyte DNA treated in vitrowith SssI methyltransferase (New England Biolabs), and a control withoutDNA. Ten μl of each MSP reaction were directly loaded onto 2% agarosegel and visualized under UV illumination. For sequencing of sodiumbisulfite-converted DNA, PCR products were amplified and cloned usingthe TOPO-TA cloning kit (Invitrogen, Breda, the Netherlands). Singlecolonies were picked and sequenced using an automated sequencer (AppliedBiosystems, Foster City, Calif.). Primer sequences used are SEQ ID NO:569 5′-GATYGGGGTGTTTTTTAGGTTT-3′ (sense primer) and SEQ ID NO: 65′-CRAACAACCAAAAACCCCTC-3′ (antisense primer).

Quantitative MSP

Quantitative real-time MSP was performed using a 7900HT real-time PCRsystem (Applied Biosystems). 2.4 μl of the modified DNA (equivalent to2.5 μg unconverted DNA) was added to a PCR mix (total volume 12 μl)containing buffer (16.6 mM (NH4)2SO4, 67 mM Tris (pH 8.8), 6.7 mM MgCl2,10 mM β-mercaptoethanol), dNTPs (5 mM), forward primer (6 ng), reverseprimer (18 ng), molecular beacon (0.16 μM), BSA (0.1 μg), and JumpstartDNA Taq polymerase (0.4 units; Sigma Aldrich). The PCR program was asfollows: 5 minutes 95° C., followed by 45 cycles of 30 seconds 95° C.,30 seconds 57° C., and 30 seconds 72° C., followed by 5 minutes 72° C.Primer sequences used are SEQ ID NO: 17 5′-GTATTTTAGTCGCGTAGAAGGC-3′(forward primer), SEQ ID NO: 18 5′-AATTTAACGAATATAAACGCTCGAC-3′ (reverseprimer) and SEQ ID NO: 195′-FAM-CGACATGCCCGAACGAACCGCGATCCCTGCATGTCG-3′-DABCYL (molecularbeacon). A standard curve (2×10⁶⁻²⁰ copies) was included to determinecopy numbers of unknown samples by interpolation of their Ct values tothe standard curve.

Loss of Heterozygosity Analysis

Allelic status was analyzed by PCR amplification with specific primerpairs flanking polymorphic microsatellite loci. The fluorescentdye-labeled microsatellite markers DS16S3089 (forward primer: SEQ ID NO:526 AGCCCTGCCTGATGAA; reverse primer: SEQ ID NO: 527 TGTGTGGGTAGCACCAA)and DS16S3071 (forward primer: SEQ ID NO: 528 AGCTCTCTGATGGGCAGTG;reverse primer: SEQ ID NO: 529 TGGAAGATAGCCCCCAAAT) located on 16q21-22were selected from genome public database. DS16S3089 is situated 1.9 Mbdownstream of NDRG4 and DS16S3071 1.8 Mb upstream of NDRG4. Matchedtumor/normal DNA samples were amplified by PCR in a 15 μl volumecontaining 0.25 mM dNTP, 0.3 μM primers, 1.5 mM MgCl2 and 0.04 unitsTaqpolymerase (platinum, Invitrogen) using 50 ng DNA as template. Thereaction mixture was subjected to 3 min of denaturing at 95° C. and 30cycles of 95° C. for 1 min, 60° C. annealing temperature for 1 min and72° C. for 1 min followed by a final extension step at 72° C. for 10min. PCR products were sequenced using an automated sequencer (AppliedBiosystems, Foster City, Calif.) and analyzed using Genemapper softwareversion 4.0 (Applied Biosystems). Only genotypes demonstrating twodifferent sizes, i.e. heterozygous MS alleles, were used for evaluatingallelic status. The allelic ratio was calculated as (N1/N2)/(T1/T2) forthe ratio of area values of tumor (T) versus the normal (N) alleles. LOHwas defined as an allelic ratio more than 1.35 and less than 0.67.

Mutation Analysis

The NDRG4 coding exons and their flanking intronic regions wereindividually amplified using genomic DNA extracted from paraffineembedded colonic adenocarcinoma tissue. Mutation analysis was examinedusing the nested PCR approach. The outside PCR was performed with 125 nggenomic DNA, 50 pmol of each forward and reverse primer and 1 units ofTaqPolymerase mixture (Invitrogen). DNA amplification was done on athermal cycler using Thermo-Fast 96-well plates (Corning) starting withan initial denaturation step at 95° C. for 3 min, followed by 35 cyclesof denaturation at 95° C. for 30s, annealing with an specifictemperature for each primer for 30s and extension at 72° C. for 30 sec.An additional final extension of 72° C. for 5 min was added. Followingthe outside PCR an inside PCR was done using the same conditions as theoutside PCR. PCR primer sets for each exon, including intron-exonboundary, are provided in detail in supplemental table 3. DNA waspurified using the Millipore multiscreen 96 wells plate (Millipore). PCRproducts were amplified using the BigDye® Terminator v1.1 Cyclesequencing kit and amplified products were sequenced using an ABI 3730DNA Analyzer (Applied Biosystems, Foster City, Calif.).

TABLE 46 NDRG4 mutation analysis primer sequences and PCR conditions.SEQ SEQ Exon ID ID Annealing No. Primer NO Sense primer NOAntisense primer temperature  2 Outside 530 CCCAGCCCCGACTTGC 531CTAAGACCTCAAAGGC 56 GCG Inside 532 TGTCCTTCTCCGCCCGG 531CTAAGACCTCAAAGGC 62 GCG  3 Outside 533 CCCCTCTGTTTGCCTTCC 534CTGGCCAGGTGGGGTG 56 Inside 533 CCCCTCTGTTTGCCTTCC 535 GCCAGGTGGGGTGAGG62 G  4 Outside 536 CTGCGTCACCTCATTCCC 537 TCACCGCTCTGGCTGA 56 TG Inside538 GAGGAGCCAAGAGCGGAG 537 TCACCGCTCTGGCTGA 62 G TG  5 Outside 539CCCCTCTGCTCAGCCATA 540 GCTGGAGACAGGCAGA 56 G GGG Inside 539CCCCTCTGCTCAGCCATA 541 GGAGACAGGCAGAGGG 56 G GG  6 Outside 542GTAGGTACCCTGAGCCCC 543 ACCCCTGGGCCCTAGC 56 C Inside 544CCCTCTGCCTGTCTCCAG 543 ACCCCTGGGCCCTAGC 62 C  7 Outside 545GGAAATGGCACCCCTAGC 546 GGGGGCATGGGGAGAC 56 Inside 547 GCACCCCTAGCCCTAGAG546 GGGGGCATGGGGAGAC 56 T  8 Outside 548 CCTTGAAGACTTTACAGA 549GTATACCCACCCCACC 56 GTGTTTC CC Inside 550 CTGCACCCATCCTGGCC 549GTATACCCACCCCACC 62 CC  9 Outside 551 GGGGTGGGGTGGGTATAC 552GCTGGGAGGGGCAAAT 56 C Inside 551 GGGGTGGGGTGGGTATAC 553 GGCAAATCCCAGATCA62 CCC 10 Outside 554 GCCTCCATCCATCTCCCT 555 GGCTGCTGATCCCACC 56 G CInside 556 CATGCCTCCATCCATCTC 555 GGCTGCTGATCCCACC 62 C C 11 + Outside557 CACCTCTGCCTCTGCCCC 558 CCCCAGTGAGCCCACA 56 12 GC Inside 559CCTCTGCCCCTCCCCC 558 CCCCAGTGAGCCCACA 62 GC 13 Outside 560TGCCTTGGCAATGGGG 561 CAGGGCTGGGGAAGAA 56 AG Inside 562 CTTGGCAATGGGGGTGG561 CAGGGCTGGGGAAGAA 62 AG 14 + Outside 563 GGAGCTTGTCCTGGAGTG 564GTGGGGTGGAAATGTA 56 15 AG CTCAC Inside 565 TGGAGTGAGGGCCCTGC 564GTGGGGTGGAAATGTA 62 CTCAC 16 Outside 566 TGCCCGCCAGTCCTCAG 567TAAAGGGAACATGAGC 56 CGG Inside 568 CAGTCCTCAGGCCCATCC 567TAAAGGGAACATGAGC 56 CGG *Number of cycles in each case was 35Quantitative Reverse Transcriptase PCR

Total RNA from cell lines, normal mucosa and tumor tissue was isolatedusing the Rneasy Mini kit (Qiagen) following the manufacturersinstructions. Possible genomic DNA contaminations were removed by DNAsetreatment with the RNase-free DNAse set (Qiagen). cDNA synthesis usingthe Iscript cDNA synthesis kit (Bio-Rad) was performed. Quantitativereal-time (RT-PCR) was performed using SYBR Green PCR master mix(Applied Biosystems, Nieuwekerk a/d IJssel, The Netherlands). RealtimeRT-PCR mixes were composed of 1×iQ SYBR Green Supermix (Bio-Rad), 400 nMof the forward (SEQ ID NO: 3 5′-GGCCTTCTGCATGTAGTGATCCG-3′) and reverse(SEQ ID NO: 4 5′-GGTGATCTCCTGCATGTCCTCG-3′) primer and cDNAcorresponding to 30 ng total RNA per reaction. As standard control,primers targeted against cyclophilin A were used. Reactions were runusing the iCycler (Bio-Rad) for 40 cycles at a Tm of 60° C. Thecomparative Ct method was used to calculate differences in mRNAexpression. To do so, the Ct value of each sample was normalized to thereference gene ([delta]Ct=Ct,sample−Ct,cyclo). Next, the fold differencein expression was calculated as 2−^([delta][delta]Ct), with [delta][delta]Ct=[delta]Ct,sample1−[delta]Ct,control.

Immunohistochemistry

Immunohistochemistry was performed on formalin-fixed, paraffin embeddedtissue sections (5 m) of normal colon mucosa and CRC tissue. Sectionswere deparaffinized in xylene, rehydrated and incubated with 1% methanolfor 30 minutes to inactivate the endogenous peroxidase. After blocking,sections were stained with the NDRG4 monoclonal antibody (AbnovaCorporation), 1:6000 diluted in Tris-buffered saline (TBS) with 0.1%Tween and 0.5% bovine serum albumin (BSA) and incubated for 60 minutes.Sections were incubated with the secondary antibody poly-HRP-GAM/R/R IgG(Immunologic, Immunovision Technologies) and staining was visualized asa brown precipitate using DAB substrate chromogen (Dako) followed byhaematoxylin counterstaining. Sections incubated without the primaryantibody served as a negative control.

Data Analysis

We used the Pearson's χ² or Fisher's Exact test and the One-way ANOVA,Kruskal-Wallis or Mann-Witney test where appropriate to comparenon-parametric and categorical data respectively. Paired samples withinthe group of cases were analyzed using the McNemar test and the pairedT-test to compare non-parametric and categorical data respectively.Logistic regression analysis was used to compare categorical dataadjusted for age and location of the tissue since significantdifferences in age and location of the different tissues were observedbetween CRC cases and controls. All quoted p-values are two-sided, and ap-value 0.05 or lower was considered statistically significant. Allstatistical tests were corrected for multiple comparisons using theBonferroni method. Data analysis was done using SPSS software (version12.0.1).

Results

NDRG4 Promoter Methylation and Expression in CRC Cell Lines

The structure of the NDRG4 gene shows a dense CpG island (GCcontent >60%, ratio of observed CpG/expected CpG >0.6 and minimum length200 bp (Gardiner-Garden and Frommer 1987)) located −556 to +869 relativeto the transcription start site as shown in FIG. 10. To assay thisregion for potential methylation we designed two different MSP primerpairs (1 and 2) amplifying overlapping fragments in the CpG island.These primers were initially used to investigate eight CRC cell lines(LS174, HCT116, HT29, RKO, CACO2, COLO2, SW48 and SW480) for DNAmethylation. All cell lines except SW480 were methylated as analyzed byMSP using both primer pairs as shown in FIG. 11a . To furtherinvestigate the pattern of CpG island methylation we performed sodiumbisulfite sequencing of HCT116 and SW480. The promoter region spanning39 CpG sites was PCR-amplified using sodium bisulfite-modified genomicDNA as template and six clones of each cell lines were sequenced.Bisulfite sequencing confirmed MSP data in that HCT116 showed almostcomplete methylation at 39 sites as depicted in FIG. 11b , whereas SW480showed almost no methylated CpG sites. Endogenous NDRG4 mRNA levels inCRC cell lines HCT116 and RKO were significant increased after treatmentwith DAC (FIG. 11c ).

Methylation of NDRG4 in Normal and CRC Tissue

Methylation of NDRG4 was confirmed in three pairs of primary tumors andmatched normal colonic mucosa by sodium bisulfite sequencing. Theresults depicted in FIG. 12a show dense methylation of the three tumorsamples while almost no methylation was observed in the normal colonmucosa. Interestingly, the density of methylation was higher in theupstream region of the NDRG4 CpG island when compared to more downsteamregion as shown in FIG. 12 a.

Subsequently, the methylation status of NDRG4 was investigated incolorectal carcinoma, adenoma and normal colorectal mucosa using twodifferent primer pairs (1 and 2). The methylation frequenties using bothprimer pairs are depicted in table 47. A significant difference (table47, p=0.042 10-7) was observed in methylation frequencies in normalmucosa of the control group (2/48 (4%)) compared to cancer tissue of CRCpatients (71/83 (86%)) using primer pair 2. In addition, we comparedNDRG4 promoter methylation in adjacent normal mucosa tissue of CRCpatients (9/78 (12%)) and the normal mucosa of non-cancerous patients(2/48 (4%) but did not find a significant difference among these twogroups (table 47). Furthermore, to investigate NDRG4 methylation inpremalignant lesions, we compared adenomas obtained from CRC patientsthat developed synchronously or metachronously to the tumour andadenomas obtained from patients that did not develop CRC after 10 yearsof follow-up. We observed a higher prevalence of NDRG4 methylation inadenomas from CRC patients although these differences did not reachstatistical significance (table 47).

TABLE 47 Methylation frequencies (%) of normal, adenoma, carcinomatissue from CRC patients and normal, adenoma tissue of non-cancerouspatients. Methylation differences are analyzed by logistic regressionadjusted for age (NDRG4p1, p2) and location (NDRG4 p1) CarcinomaControls Normal tissue Adenoma tissue tissue normal P controls CRC+ PControls CRC+ P NDRG4 71% 0%  0.02 × 10⁻² 0%  3% NS 13% 41% NS p1 NDRG486% 4% 0.042 × 10⁻⁷ 4% 12% NS 55% 66% NS p2Abbreviations: CRC+, colorectal cancer patients; P, P-value; NS, notsignificant

To confirm the high prevalence of NDRG4 promoter methylation in CRC, weanalyzed a second independent series of 183 CRC samples. Comparable tothe results of the first study series we observed that 70% (127/183) ofCRC patients presented NDRG4 methylation.

Further analysis of the clinicopathologic features of patients withprimary CRC with regard to NDRG4 promoter methylation did not reveal anyassociation with age at diagnosis, sex, location of the tumor or the TNMstage for both independent series using primer 2 (table 49). Toinvestigate NDRG4 promoter methylation during cancer progression wecompared the frequency of methylation from normal mucosa to adenoma andcarcinoma tissues in patients for which all the three tissues wereavailable (table 48). Our results show that NDRG4 is significantly(table 48, p<0.02 10−2) more frequently methylated in carcinomas (84%)compared to normal mucosa adjacent to the tumor (16%). In addition tothe carcinomas, adenoma samples from CRC patients also exhibitsignificantly (table 48, p<0.03 10−3) higher NDRG4 methylationfrequencies (61%) compared to normal colon samples (14%). Finally, NDRG4methylation was increased in carcinoma tissues (81%) compared to adenomasamples (63%) although this enhancement was not significant (primer pair2, table 48).

TABLE 48 NDRG4 Methylation frequencies (%) of carcinoma tissue, adenomaand normal tissue from colorectal cancer patients. Methylationdifferences were analyzed by Mc Nemar test. CRC Normal Adenoma NormalCarcinoma Adenoma Carcinoma patients tissue tissue P tissue tissue Ptissue tissue P NDRG  0% 34% 0.003  0% 73%  0.01 × 10⁻⁴ 39% 76% 0.012 4p1 NDRG 14% 61% <0.03 × 10⁻³ 16% 84% <0.02 × 10⁻² 63% 81% NS 4 p2

Frequencies may vary because of missing data for some variables.

Abbreviations: CRC+, colorectal cancer patients; P, P-value; NS, notsignificant

The different series were analyzed using two different primer pairs 1and 2 amplifying overlapping fragments in the CpG island, as depicted inFIG. 10. Using primer pair 1 we observed overall the same resultscompared to primer pair 2 however we found an increase of NDRG4methylation for all the subgroups using primer pair 2 compared to primerpair 1. Interestingly, we found a significant difference (table 2,p=0.012) in promoter methylation in adenomas of CRC patients (55/77(41%)) compared to the carcinomas (55/77 (71%)) which was not observedusing primer pair 2. In addition, comparing the NDRG4 methylation statusof adenomas obtained from CRC patients that developed synchronously ormetachronously to the tumour (24/58(41%)) and adenomas obtained frompatients that did not develop CRC (4/31 (13%) we observed a enormousincrease of NDRG4 methylation in adenomas from CRC patients using primerpair 1 although these differences also did also not reach statisticalsignificance. Further analysis of the clinicopathologic features ofpatients with primary CRC with regard to NDRG4 promoter methylation forboth independent series did not reveal any association with age atdiagnosis, sex or the TNM stage. However, we did find a significantcorrelation between promoter methylation and the location of the tumorusing primer pair 1 (table 49, p=0.034).

TABLE 49 Prevalence (%) of promoter methylation of NDRG4 in relation toclinicopathological features of carcinoma tissue for two independentseries. Methylation differences were analyzed by chi-square %methylation % % NDRG4p2 methylation methylation IndependentCharacteristics NDRG4p1 NDRG4p2 series TNM stage* I 15% 16% 23% II 33%32% 33% III 40% 41% 30% IV 13% 11% 13% P NS NS NS Tumor Location‡proximal 65% 56% 37% distal 35% 44% 63% P 0.034 NS NS Sex* Male 42% 48%55% Female 58% 52% 44% P NS NS NS Age at diagnosis§ <=mean 40% 48%48% >mean 60% 52% 52% P NS NS NS Abbreviations: P, P-value; NS, notsignificantNDRG4 Promoter Methylation in Other Neoplasias

Next, we asked whether NDRG4 promoter methylation is present in othertumor tissues. Therefore 119 primary tumor specimens covering 7different tumor types were analyzed using MSP primer pair 2. No orlittle methylation was found in skin (0/8, 0%), kidney (1/10, 10%),ovary (0/20, 0%), prostate (0/10, 0%) and breast (lobular (0/7, 0%) andductal (0/9, 0%)) carcinomas. In contrast, NDRG4 promoter was frequentlymethylated in adenocarcinomas of the esophagus (13/16, 81%), while nomethylation was found in esophageal squamous cancers (0/12, 0%). Bothdiffuse type (8/11, 73%) and intestinal type (9/11, 82%) carcinomas ofthe stomach were frequently methylated while the normal mucosa of thestomach did not show any methylation (0/5, 0%).

NDRG4 Promoter Methylation in Fecal DNA

The high prevalence of NDRG4 promoter methylation in CRC and the absenceof methylation in normal colon mucosa suggest that NDRG4 promotermethylation could be a sensitive and specific biomarker for non-invasivedetection of CRC. Therefore, we developed a quantitative MSP assay usingmolecular beacon technology and analyzed fecal DNA of 21 CRC patientsand 67 healthy controls. NDGR4 promoter methylation could be detected in16/21 CRC patients yielding a 76% sensitivity for the detection of CRC.Only 2/67 (3%) of healthy controls tested positive for NDRG4methylation, which resulted in a clinical specificity for the assay of97%. Stool samples were obtained from CRC patients covering alldifferent TNM stages. The assay had a 75% sensitivity among CRC patientswith early stage colon cancer (stage I and II) and 80% of sensitivityamong later stage patients (stage III and IV).

NDRG4 RNA and Protein Expression

To analyse whether methylation of the promoter CpG island of NDRG4 isassociated with gene silencing we investigated mRNA expression of NDRG4in CRC cell lines, three pairs of CRC tissues and matching normal colonmucosa. In all three CRCs, mRNA levels were significantly downregulated(97, 70% and 98% respectively) when compared to normal colon mucosa(FIG. 12b ).

To investigate the protein expression of NDRG4 in both normal colonicmucosa and colon cancers, we performed NDRG4 immunohistochemistrydemonstrating the presence of NDRG4 protein expression in the cytoplasmof normal colon mucosa while protein expression is lost in half of CRCs(FIG. 12c ). Subsequently, we performed immunohistochemical analysis ofNDRG4 expression on 19 CRC samples. Eleven of these patients had amethylated NDRG4 promoter. However, we could not find a significantassociation between NDRG4 promoter methylation and NDRG4 expression(data not shown). This observation suggests that other mechanisms mightlead to NDRG4 inactivation.

Loss of Heterozygosity and Mutation Analysis of the NDRG4 Gene in CRC

Macrodissected CRC tissue and corresponding normal tissues of 86 CRCpatients were analyzed using the microsatellite markers DS16S3089 andDS16S3071. The two markers showed a heterozygosity of 77.4% and 35.4%respectively. Of these, 59 cases were informative; 18 tumors (30.5%)showed LOH with at least one marker on chromosome 16q.

Twelve primary CRC and CRC cell lines HCT116 and SW480 were analyzed forNDRG4 mutations. No inactivating mutations within the coding region ofthe NDRG4 gene were detected in 12 colorectal carcinomas. However, wefound one novel nonsynonymous mutation in the SW480 cell line (40662A→AGIle65Val). As part of the mutational analysis, 2 previously reportedSNPs (NCBI SNP database) were detected. One SNP was observed in 1/12 CRCpatients (43760G→GG Va1224Val refSNP rs 17821543). The second SNP wasobserved in 9/12 CRC patients (48311A→AG Ser354Ser refSNP rs 42945).

Discussion

The progression of CRC from small benign colorectal adenomas to largerand more dysplastic lesions takes several decades and identifying earlystages would improve management and treatment of this disease (Brennerand Rennert 2005). Colonoscopy is currently the best technique fordetecting CRC or its precursor lesions from the age of 50 years onwards.Testing for the presence of fecal occult blood (FOBT) as preselectionfor colonoscopy is the only non-invasive screening method with proveneffectiveness, reducing both the incidence and the risk of death fromCRC when used programmatically.

However, both sensitivity and specificity of FOBT is low and thereforethere is an urgent need for more sensitive and specific non-invasivescreening tests. A promising option is analyzing (expression of)cancer-specific molecules such as DNA, RNA and protein in blood andtissue. First attempts to detect genetic alterations are promising(Dong, Traverso et al. 2001; Traverso, Shuber et al. 2002) althoughstill need improvement. Markers of choice have been TP53, K-ras and APCmutations and in addition BAT-26 instability and long DNA (a marker fornon-apoptotic shedding of epithelial colonocytes). Recently, CpG islandhypermethylation can also be used as a (prognostic) marker fornon-invasive detection of CRC in different biological samples (Esteller2003; Chen, Han et al. 2005; Ebert, Model et al. 2006). Over the lastyears, several genes have been described to be methylated in CRC usingdifferent techniques.

Here we used MSP, quantitative MSP and bisulfite sequencing to analyseNDRG4 as a biomarker for the early detection of colorectal and othergastrointestinal cancers. (ARRAY) The NDRG4 promoter CpG island wasdemonstrated to be methylated in two independent large series of CRCcases. In the first series we included normal mucosa of non-cancerouspatients since the normal mucosa from the CRC patients is situatedwithin the same bowel segment as the tumor and can be contaminated withmalignant cells or a field-effect could have change the molecularsignature of this cell as described for MGMT (issa, 2005). Nevertheless,by performing statistical analysis we could not find any significantlydifference in methylation between these two groups. Chronic inflammationhas previously been shown to accelerate DNA methylation in normaltissues (Issa, Ahuja et al. 2001).

Therefore additional screens with inflamed colon mucosa are expected ina screening setting. In our study population, inclusion of inflamedmucosa to the normal mucosa of control patients slightly reduced thespecificity of NDRG4 from 96% to 94%. Because we found a difference inthe density of methylation in the promoter area of NDRG4 by bisulfitesequencing, we used two different primer sets to investigated themethylation status of NDRG4. Interestingly, using primer pair 2, wefound 86% of methylation in carcinoma tissue while only 71% was observedby use of primer pair 1. This increased detection of methylation usingprimer pair 2 was observed for all the subgroups of this series as shownin table 47. Primer pair 2 is situated more to the 5′region of the gene.The frequencies of methylation were lower near the transcription startsite. We hypothesize that NDRG4 hypermethylation initially occurs at the5′ end of the NDRG4 CpG island and spreads towards the transcriptionstart site before ultimately shutting down NDRG4 mRNA expression, as hasalso been observed for RUNX3 (Turker 2002; Homma, Tamura et al. 2006).In addition, we found a significant difference (p=0.012) in methylationfrequency using primer pair 1, between adenoma tissue and carcinomatissue within the group of CRCs. Therefore, we speculated, thatspreading of DNA methylation in the promoter area of NDRG4 towards thetranscription start site occurs during cancer progression.

Remarkably, using primer pair 1, hypermethylation was more frequentlypresent in progressed adenomas from the CRC patients (41%) when comparedto the non progressing adenomas of the CRC-patients (13%). The capacityto distinguish adenomas that progress to cancer from those that will notprogress is highly important for CRC screening (Hermsen, Postma et al.2002). Whereas this difference can not be made macroscopically,endoscopic screening strategies aiming to detect and remove all adenomaswill be inherently unspecific. The majority of adenomas removed wouldnot have progressed to cancer because only a small percentage of thesebenign precursor lesions will progress into a carcinoma (Lengauer,Kinzler et al. 1998). These data might indicate that NDRG4 promotermethylation in adenoma tissue (in the region we investigated) is apossible risk factor for developing a colon tumor.

Recently, it has been reported that promoter methylation can be detectedin biological fluids such as blood, urine or stool and may allow earlydiagnosis of various cancers, including CRC. Some studies have shownthat methylation of one gene promoter can be used as a screening methodfor fecal DNA methylation detection. For example, promoter methylationof SFR2, Vimentin and HIC1 can be detected in fecal DNA of CRC patientswith a sensitivity of 77%, 43% and 42% respectively and a specificity of77%, 90% and 95% respectively (Muller, Oberwalder et al. 2004; Chen, Hanet al. 2005; Lenhard, Bommer et al. 2005). NDRG4 methylation in fecalDNA as a single marker can differentiate cancer from controls with asensitivity of 76% and a specificity of 97%.

In order to be a specific biomarker for CRC, analysis of tissuespecificity was performed; we found NDRG4 methylation in other tumors ofthe gastrointestinal tract, namely oesophagus and gastric cancers. Thisdata indicate that methylation of NDRG4 may serve as a marker for othergastrointestinal tumors as well.

We next studied whether methylation of NDRG4 is associated withdownregulation of NDRG4 RNA and protein expression. So far, theexpression of NDRG4 has only been documented in the brain and heart byuse of Northern blotting. We observed expression of NDRG4 in normalcolon tissue and downregulation in al three tumor tissues. Subsequently,we performed immunohistochemical analysis of NDRG4 expression on 19 CRCsamples from the CRC patients for which paraffin-embedded tissues wereavailable. Eleven of these patients had a methylated NDRG4 promoter.However, we could not find a significant association between NDRG4promoter methylation and NDRG4 expression. Some tumors had a methylatedNDRG4 promoter although still expressed NDRG4 protein. The methylationthat we detected using MSP might reflect methylation of only a fewcancer cells or methylation of only one of two NDRG4 alleles (andabsence in the other). Nevertheless, some tumors lack expression ofNDRG4 protein while no promoter methylation was observed. Thisobservation suggests that other mechanisms might lead to NDRG4inactivation. No mutations were found, indicating that mutationalinactivation of the NDRG4 gene might not play a mayor role in CRC. Ourresults confirmed previous data on NDRG4 mutation studies (Sjoblom,Jones et al. 2006). However, LOH at 16q is seen in about 30% of the CRCcases. Frequent LOH of 16q had previously been described in a widevariety of solid tumor types as breast (Rakha, Green et al. 2006), liver(Sakai, Nagahara et al. 1992; Bando, Nagai et al. 2000), prostate (Elo,Harkonen et al. 1997), ovarian (Kawakami, Staub et al. 1999) and Wilms'tumors (Mason, Goodfellow et al. 2000) but until now it has not beendescribed in CRC. Because NDRG4 is downregulated in most of the coloncancer cells compared to normal colonic epithelial cells we hypothesesthat NDRG4 has a tumor suppressor function in cancer.

In conclusion, we are the first group who described a role for NDRG4 incancer and our data indicate that NDRG4 is a potential novel marker forCRC with a very high sensitivity and specificity of 76% and 100%respectively. Although the sensitivity and specificity of NDRG4 as amarker alone is already very high, the diagnostic accuracy of NDRG4 maybe enhanced by the addition of other markers analyzed in patients withCRC as well. This may augment the ability to identify patients withcancer in a multipanel methylation-based diagnostic test.

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Plasmid material corresponding to a promoter region of NDRG4 and OSMRgene was used to test additional assay designs. The plasmid for thestandard curve was generated as follows: the promoter sequence asdefined by the primers is PCR amplified and cloned (using suitableisolated and bisulphite modified cell line DNA). The sequence isverified by sequencing and compared to the published promoter sequence.A serial dilution of either NDRG4 or OSMR plasmid material (2×10⁶ to2×10¹copies/5 μl) was loaded in duplicate. 5 μl of plasmid dilution orbuffer (non template control) was added to a 20 μl PCR mix containingthe specified primer and beacon detector sequences as previouslydescribed. Results were generated using the SDS 2.2 software fromApplied Biosystems with automatic baseline and threshold settings. Datawere exported as Ct values (cycle number at which the amplificationcurves cross the threshold value, set automatically by the software).

NDRG4

Initial real-time results for 2 different NDRG4 assay designs arepresented in table 50 and 51. The primer and beacon combinations usedfor the respective assays NDRG4_1a and NDRG4_1b were previouslydescribed. Underscore 1a and 1b reflect the different primer and/orbeacon combinations used for assessing the methylation status of theNDRG4 gene. NDRG4_1a corresponds to the preferred NDRG4 assay design,also simply referred to as NDRG4 (see Table 4). Comparable results wereobtained for both assay designs. Clinical sample data provided in thisinvention are generated using the preferred NDRG4 assay design(=NDRG4=NDRG4_1 a)

TABLE 50 Realtime MSP results obtained for NDRG4_1a assay on plasmidmaterial. Resulting standard curve (y = −3.3321x + 39.862; R2 = 0.9991)corresponds to a PCR efficiency of 100%. Log Duplicate Average AssayTask Ct Quantity copies Ct Ct ΔCt NDRG4_1a Standard 18.82 2000000 6.3018.92 18.87 0.09 NDRG4_1a Standard 22.09 200000 5.30 22.22 22.15 0.13NDRG4_1a Standard 25.42 20000 4.30 25.47 25.45 0.06 NDRG4_1a Standard28.86 2000 3.30 28.94 28.90 0.08 NDRG4_1a Standard 32.58 200 2.30 32.4832.53 0.10 NDRG4_1a Standard 34.92 20 1.30 35.64 35.28 0.71 NDRG4_1a NTCUnde- 0 Unde- Undeter. Undeter. termined termined

TABLE 51 Real time MSP results obtained for NDRG4_1b assay on plasmidmaterial. Resulting standard curve (y = −3.4181x + 40.991; R2 = 0.9991)corresponds to a PCR efficiency of 99.2%. Log Duplicate Average AssayTask Ct Quantity copies Ct Ct ΔCt NDRG4_1b Standard 19.48 2000000 6.3019.59 19.53 0.12 NDRG4_1b Standard 22.93 200000 5.30 22.92 22.92 0.01NDRG4_1b Standard 26.26 20000 4.30 26.18 26.22 0.08 NDRG4_1b Standard29.65 2000 3.30 29.67 29.66 0.02 NDRG4_1b Standard 32.82 200 2.30 32.8332.82 0.01 NDRG4_1b Standard 36.75 20 1.30 36.91 36.83 0.16 NDRG4_1b NTCUnde- 0 Unde- Undet Undet termined terminedOSMR

Initial real-time results for 3 different OSMR assay designs arepresented in below Tables 52 to 54. The primer and beacon combinationsused for the respective assays OSMR_1, OSMR_3 [=OMSR (3)] and OSMR_4[=OSMR (4)] were previously described. Underscore 1, 3 and 4 reflect thedifferent primer and/or beacon combinations used for assessing themethylation status of the OSMR gene. Comparable results were obtainedfor all three assay designs.

TABLE 52 Real time MSP result obtained for OSMR_1 assays on plasmidmaterial. Resulting standard curve (y = −3.3326x + 41.136; R2 = 0.9993)corresponds to a PCR efficiency of 99.6%. Log Duplicate Average AssayTask Ct Quantity copies Ct Ct ΔCt OSMR_1 Standard 20.04 2000000 6.3020.14 20.09 0.09 OSMR_1 Standard 23.48 200000 5.30 23.41 23.44 0.07OSMR_1 Standard 26.73 20000 4.30 26.85 26.79 0.12 OSMR_1 Standard 30.132000 3.30 30.26 30.19 0.13 OSMR_1 Standard 33.55 200 2.30 33.93 33.740.38 OSMR_1 Standard 36.54 20 1.30 36.58 36.56 0.04 OSMR_1 NTC Undeter-0 Undeter- Undet Undet mined mined

TABLE 52 Real time MSP result obtained for OSMR_3 assays on plasmidmaterial. Resulting standard curve (y = −3.3909x + 38.398; R2 = 0.9999)corresponds to a PCR efficiency of 97.2%. Log Duplicate Average AssayTask Ct Quantity copies Ct Ct ΔCt OSMR_3 Standard 16.93 2000000 6.3017.16 17.04 0.23 OSMR_3 Standard 20.41 200000 5.30 20.29 20.35 0.12OSMR_3 Standard 23.97 20000 4.30 23.83 23.90 0.14 OSMR_3 Standard 27.222000 3.30 27.16 27.19 0.06 OSMR_3 Standard 30.51 200 2.30 30.67 30.590.16 OSMR_3 Standard 34.18 20 1.30 33.77 33.98 0.41 OSMR_3 NTC 38.13 0Undeter- Undet Undet mined

TABLE 54 Real time MSP result obtained for OSMR_4 assays on plasmidmaterial. Resulting standard curve (y = −3.2795x + 38.77; R2 = 0.9997)corresponds to a PCR efficiency of 100.8% Log Duplicate Average AssayTask Ct Quantity copies Ct Ct ΔCt OSMR_4 Standard 18.24 2000000 6.3017.90 18.07 0.33 OSMR_4 Standard 21.56 200000 5.30 21.05 21.31 0.51OSMR_4 Standard 24.79 20000 4.30 24.64 24.72 0.15 OSMR_4 Standard 28.242000 3.30 27.91 28.08 0.33 OSMR_4 Standard 31.37 200 2.30 31.18 31.280.19 OSMR_4 Standard 34.63 20 1.30 34.12 34.37 0.50 OSMR_4 NTC Undeter-0 Undeter- Undet Undet mined mined6) Testing and Validation of Further CRC Markers in Bodily Fluid TestSamples

New markers added: BNIP3, FOXE1, JAM3, PHACTR3, TPFI2, SOX17 and SYNE1(and also JPH3 stool data). Suitable primers and probes for determiningthe methylation status of these genes are set forth in Tables 12 (and 13to 18) above.

Methods and Results

Clinical Samples

Samples were collected from centers in Germany and The Netherlands

TABLE 55 Samples for DNA extraction from plasma (blood origin) Sampletype Numbers Normal 10 Colorectal Cancer stage III 6 Colorectal Cancerstage IV 4

TABLE 56 Samples for DNA extraction from stool Sample type NumbersControl (Normal) 7 Case (Colorectal Cancer) 1 Case (Colorectal Cancer) 6Marker Testing on Clinical Samples

Experiments were performed as previously described. Briefly DNA wasextracted from stool and/or plasma followed by bisulfite treatment.Samples were tested by real-time MSP assays, using 384 well plates witha 12 μl final volume. The template volume is 2.4 μl with a mix volume of9.6 μl. Results were generated using the SDS 2.2 software (AppliedBiosystems), exported as Ct values (cycle number at which theamplification curves cross the threshold value, set automatically by thesoftware). Copy numbers are extrapolated using a standard curve.

The individual performance of the 8 gene assays TFPI2, BNIP3, FOXE1,SYNE1, SOX17, PHACTR3, JAM3 and JPH3 in plasma and stool samples isshown in Table XV (except for JPH3: stool data only). Sensitivity valuesfor plasma and stool are ranging from 30 to 70% and 0 to 57%respectively with a corresponding specificity of a 100%. When optimizingfor sensitivity, 80% sensitivity for TFPI2 and 50% sensitivity forPHACTR3 is obtained in plasma samples with a corresponding specificityof 90%. It is observed that for some markers (TFPI2, BNIP3, FOXE1, SYNE1and SOX17) sensitivity of colorectal cancer detection is higher whenusing plasma samples compared to stool samples.

TABLE 57 Individual gene performance: Sensitivity and specificity ofTFPI2, BNIP3, FOXE1, SYNE1, SOX17, PHACTR3, JAM3 markers on stool andplasma samples. Sensitivity and specificity results for the JPH3 markerwere only obtained for stool using this sample set, plasma data wereenabled earlier with a different sample set. optimized for Sensitivityoptimized for Specificity Sensi- cut Specificity Sensitivity cutoffSpecificity tivity off TFPI2 Stool 100 57 10 TFPI2 Plasma 100 70 1 90 800 BNIP3 Stool 100 0 7 BNIP3 Plasma 100 30 0 FOXE1 Stool 100 57 0 FOXE1Plasma 100 60 0 SYNE1 Stool 100 57 2 SYNE1 Plasma 100 60 0 SOX17 Stool100 57 30 SOX17 Plasma 100 60 2 PHACTR3 Stool 100 43 8 PHACTR3 100 40 290 50 0 Plasma JAM3 Stool 100 43 1 JAM3 Plasma 100 30 1 JPH3 Stool 10014 20 JPH3 Plasma see previous colon results

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Moreover, all embodiments described herein areconsidered to be broadly applicable and combinable with any and allother consistent embodiments, as appropriate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

The invention claimed is:
 1. A method, comprising: (a) hybridizing DNA from a sample to an oligonucleotide capture probe and isolating DNA hybridized to the oligonucleotide capture probe; (b) treating DNA obtained from step (a) with a reagent that modifies unmethylated DNA; (c) amplifying the treated DNA with primers that hybridize to CpG dinucleotides in a target sequence that is within a promoter region of a human NDRG4 gene that corresponds to positions 1-1000 of the methylated and bisulfite-treated sequence of SEQ ID NO: 524, to produce amplified DNA that comprises CpG dinucleotides; and (d) detecting production of the amplified DNA.
 2. The method of claim 1, wherein the reagent that modifies unmethylated DNA is selected from a methylation-sensitive restriction enzyme and a reagent that converts non-methylated cytosines to uracil.
 3. The method of claim 2, wherein the reagent that modifies unmethylated DNA is a reagent that converts non-methylated cytosines to uracil.
 4. The method of claim 1, wherein detecting production of the amplified DNA comprises detecting hybridization of a probe that hybridizes to the amplified DNA.
 5. The method of claim 4, wherein the probe hybridizes to CpG dinucleotides in the target sequence.
 6. The method of claim 4, wherein the probe is a hydrolytic probe and hybridization of the probe to the amplified DNA is detected by detecting hydrolysis of the probe.
 7. The method of claim 1, wherein the sample comprises one or more of a tissue sample, a fecal sample and/or a bodily fluid sample.
 8. The method of claim 7, wherein the bodily fluid sample comprises a blood sample and/or a plasma sample. 